Contrast agents

ABSTRACT

The invention provides a composition of matter of the formula (I): V-L-R, where V is a vector moiety having affinity for an angiogenesis-related endothelias cell receptor, L is a linker moiety or a bond and R is a detectable moiety, characterised in that V is a non-peptidic organic group, or V is peptidic and R is a macromolecular or particulate species providing a multiplicity of labels detectable in in vivo imaging.

[0001] This invention relates to diagnostic imaging techniques in whicha disease state may be imaged using a targeted contrast agent and totargeted contrast agents suitable for use in such techniques. Moreparticularly the invention relates to the use of such contrast agents inwhich the targeting vector binds to receptors associated withangiogenesis. Such contrast agents may thus be used for diagnosis of forexample malignant diseases, heart diseases, inflammation-relateddiseases, rheumatoid arthritis and Kaposi's sarcoma. Moreover suchagents may be used in therapeutic treatment of these diseases.

[0002] New blood vessels can be formed by two different mechanisms:vasculogenesis or angiogenesis. Angiogenesis is the formation of newblood vessels by branching from existing vessels. The primary stimulusfor this process may be inadequate supply of nutrients and oxygen(hypoxia) to cells in a tissue. The cells may respond by secretingangiogenic factors, of which there are many; one example, which isfrequently referred to, is vascular endothelial growth factor (VEGF).These factors initiate the secretion of proteolytic enzymes which breakdown the proteins of the basement membrane, as well as inhibitors whichlimit the action of these potentially harmful enzymes. The otherprominent effect of angiogenic factors is to cause endothelial cells tomigrate and divide. Endothelial cells which are attached to the basementmembrane, which forms a continuous sheet around blood vessels on thecontralumenal side, do not undergo mitosis. The combined effect of lossof attachment and signals from the receptors for angiogenic factors isto cause the endothelial cells to move, multiply, and rearrangethemselves, and finally to synthesise a basement membrane around the newvessels.

[0003] Angiogenesis is prominent in the growth and remodeling oftissues, including wound healing and inflammatory processes. Tumors mustinitiate angiogenesis when they reach millimeter size in order to keepup their rate of growth. As angiogenesis is accompanied bycharacteristic changes in the endothelial cells and their environment,this process is a promising target for therapeutic intervention.Inhibition of angiogenesis is also considered to be a promising strategyfor antitumor therapy. The transformations accompanying angiogenesis arealso very promising for diagnosis, an obvious example being malignantdisease, but the concept also shows great promise in inflammation and avariety of inflammation-related diseases, including atherosclerosis, themacrophages of early atherosclerotic lesions being potential sources ofangiogenic factors. These factors are also involved inre-vascularisation of infarcted parts of the myocardium, which occurs ifa stenosis is released within a short time.

[0004] Angiogenesis involves receptors which are unique to endothelialcells. The surface of these cells is remodelled in preparation formigration, and cryptic structures are exposed where the basementmembrane is degraded, in addition to the variety of proteins which areinvolved in effecting and controlling proteolysis. A number of knownreceptors/targets associated with angiogenesis are listed in Table 1below. In the case of tumors, the resulting network of blood vessels isusually disorganised, with the formation of sharp kinks and alsoarteriovenous shunts. Using the targeting principles described in thepresent disclosure, angiogenesis may be detected by the majority of theimaging modalities in use in medicine. TABLE 1 Receptors/targetsassociated with angiogenesis Receptors/Targets α₂-antiplasmin basementmembrane components basic fibroblast growth factor (bFGF) biglycan(dermatan sulfate proteoglycan) cartilage-derived inhibitor [inhibitor]CD34 collagen type I, IV, VI, VIII decorin (dermatan sulfateproteoglycan) dermatan sulfate proteoglycans endoglin endosialinendothelin epidermal growth factor (heparin-binding) fibrinfibrinopeptide B fibroblast growth factor, FGF-3, basic fibronectinFlt-1/KDR, Flt-4 (VEGF receptor) FLT-1 (fins-like tyrosine kinase)(VEGF-A receptor) heparan sulfate hepatocyte growth factor hepatocytegrowth factor receptor (c-met) hyaluronan insulin-like growth factorinsulin-like growth factor/mannose-6-phosphate receptor integrins: β₃and β₅, integrin α_(v)β₃, integrin α₆β_(1 (laminin receptor), integrin)α₆, integrin β₁, integrin α₂β₁, integrin α₅ (subunit of the fibronectinreceptor), integrin α_(v)β₅, fibrin receptors interferon-α, β[inhibitors] interleukins: IL-8, IL-12 [inhibitor] Jagged gene product.laminin laminin fragments leukemia inhibitory factor Ly-6 (a lymphocyteactivation protein) matrix metalloprotease-2 metalloproteinasesmetalloproteinase inhibitors MHC class II Notch gene product placentalgrowth factor placental proliferin-related protein plasmiflogenplasminogen activator plasminogen activator inhibitor-1 plasminogenactivator receptor platelet-derived growth factor (e.g. type BB)platelet-derived endothelial cell growth factor platelet factor 4[inhibitor] pleiotropin proliferin, proliferin-related protein receptortyrosine kinases selectins: E-selectin SPARC stress proteins (molecularcharperones) (glucose regulated, heat shock families) syndecan tissueinhibitor of metalloproteinases (e.g. TIMP-2) thrombinthrombin-receptor-activating tetradecapeptide thrombospondin [inbibitor]TIE receptors (tyrosine kinases with Ig- and EGF-like domains) tissuefactor transforming growth factor-α, β tumor growth factor-α tumornecrosis factor urokinase-type plasminogen activator receptor Vascularendothelial growth factor-A Vascular endothelial growth factor-relatedprotein Vascular endothelial growth factor-A receptor vitronectin vonWillebrand factor

[0005] As indicated above, many undesired conditions are associated withneovascularization or angiogenesis, the development or proliferation ofnew blood vessels. Examples of such conditions are listed in Table 2below TABLE 2 Diseases and indications associated with angiogenesisDiseases/Indications arteriovenous malformations astrocytomasatherosclerosis breast cancers choriocarcinomas colorectal cancersgingivitis glioblastomas gliomas hemangiomas (childhood, capillary)hepatomas hyperplastic endometrium inflammation (e.g. chronic) ischemicmyocardium Kaposi sarcoma lung cancers macular degeneration melanomametastasis neuroblastomas occluding peripheral artery diseaseosteoarthritis ovarian cancers pancreatic cancers prostate cancerspsoriasis retinopathy (diabetic, proliferative) rheumatoid arthritisscleroderma seminomas skin cancers solid tumor formation ulcerativecolitis

[0006] The surface cells, endothelial cells, of such new blood vesselshave greater than normal concentrations of various surface ortransmembrane receptors, such as for example receptor tyrosine kinases(RTK), and it has been proposed to use radiolabelled orchromophore-labelled antibodies to such receptors, or similarly labelledanalogues of natural protein ligands for such receptors, as a means ofdetecting centres of angiogenesis (see for example WO95/26364 (Orion),WO96/30046 (Genentech) and WO95/12414 (Repligen)).

[0007] Peptidic ligands however have relatively few attachment sites fordetectable labels (reporters) and attachment of reporters at many siteson such peptidic ligands will affect the conformations which the ligandmay adopt. A further problem with peptides is that they are oftenunstable in vivo.

[0008] There is therefore still a need for effective targeted contrastagents with affinities for receptors associated with angiogenesis.

[0009] The present invention addresses this need in two ways—firstly byproviding targeted contrast agents based on non-peptidic ligands(vectors)—and secondly by providing targeted contrast agents based onmacromolecular or particulate reporters providing a multiplicity ofdetectable labels (multireporters).

[0010] Thus viewed from one aspect the invention provides a compositionof matter of formula I

V-L-R  (I)

[0011] wherein V is a vector moiety having affinity for anangiogenesis-related endothelial cell receptor, L is a linker moiety ora bond, and R is a detectable reporter moiety, preferably a gas-freedetectable reporter moiety, e.g. detectable in an imaging procedure,such as in vivo imaging of the human or vascularized non-human animalbody (e.g. mammalian, avian or reptilian body), characterised in that Vis a non-peptidic organic group, or V is peptidic and R is amacromolecular or particulate species providing a multiplicity of labelsdetectable in in vivo imaging.

[0012] Where R is a macromolecular or particulate species providing amultiplicity of labels, these may be labels which individually aredetctable (e.g. paramagnetic or radioactive species) or they mayinteract to produce a detectable material, e.g. by virtue of acooperative magnetic phenomenon. Examples of such multi-reportersinclude polychelates and polyionic species, and ferromagnetic,ferrimagnetic and superparamagnetic particles.

[0013] In many instances, the composition of matter of formula I will bea characterisable compound. In others it may be a combination ofcompounds bonded or otherwise associated, eg. conjugated, with eachother. For convenience sake, the composition of matter will be referredto hereinafter as an agent.

[0014] By “gas” is meant a material or mixture of materials which isgaseous at 37° C. By “gas-free” is meant a reporter which does notcontain sufficient gas to be detectable in ultrasonography in vivo.Contrast agents comprising gas-containing reporters are described in ourcopending International Patent Application No. PCT/GB97/02958 filed Oct.28, 1997.

[0015] Viewed from a further aspect the invention provides apharmaceutical composition comprising an effective amount (eg. an amounteffective to enhance image contrast in in vivo imaging) of an agent offormula I together with at least one pharmaceutically effective carrieror excipient.

[0016] Viewed from a still further aspect the invention provides the useof an agent of formula I for the manufacture of a contrast medium foruse in a method of diagnosis involving administration of said contrastmedium to an animate subject and generation of an image of at least partof said subject.

[0017] Viewed from a still further aspect the invention provides amethod of generating an image of an animate human or non-human(preferably mammalian or avian) animal subject involving administering acontrast agent to said subject, eq. into the vascular system or the gitract, and generating an image of at least a part of said subject towhich said contrast agent has distributed, eg. by X-ray, MR, ultrasound,scintigraphy, PET, SPECT, electrical impedance, light or magnetometricimaging modalities, characterised in that as said contrast agent is usedan agent of formula I.

[0018] Viewed from a further aspect the invention provides a method ofmonitoring the effect of treatment of a human or non-human animalsubject with a drug to combat a condition associated with angiogenesis,e.g. a cytotoxic agent, said method involving administering to saidsubject an agent of formula I and detecting the uptake of said agent byendothelial cell receptors, in particular receptors in a region ofangiogenesis, said administration and detection optionally butpreferably being effected repeatedly, eg. before, during and aftertreatment with said drug.

[0019] Viewed from a yet further aspect the invention provides a processfor the preparation of an agent of formula I, said process comprisingconjugating (i) compound having binding affinity for an endothelial cellreceptor associated with angiogenesis to (ii) a compound detectable in adiagnostic imaging procedure or a chelant compound and if necessarymetallating cheiant groups in the resultant conjugate with a metal iondetectable in a diagnostic imaging procedure.

[0020] The agents of formula I have three characteristic components: avector (V); a linker (L); and a reporter (R). The vector must have theability to target the compound to a region of angiogenesis, the reportermust be detectable in an in vivo diagnostic imaging procedure; and thelinker must couple vector to reporter, at least until the reporter hasbeen delivered to the region of angiogenesis and-preferably until theimaging procedure has been completed.

[0021] Vectors

[0022] As the vector may be used any peptidic or, more preferably,non-peptidic compound having affinity for receptors associated withangiogenesis.

[0023] Non-peptidic compounds are preferably used as peptidic vectorswill generally have poor biological stability and may provoke undesiredresponses by the body.

[0024] Preferably the vector is a compound which does not elicit anyunacceptable biological response, especially one which does not actuallypromote angiogenesis.

[0025] Particularly preferably the vector is an angiogenesis inhibitor,especially preferably a non-peptidic angiogenesis inhibitor.

[0026] Examples of non-peptidic angiogenesis inhibitors are described inWO94/17084 (British Biotech), EP-A-618208 (Daiichi), WO94/13277 (ICRT),WO95/06473 (Nippon Kayaku), WO94/21612 (Otsuka), WO97/37655 (Merck),WO97/30035 (Zeneca), EP-A-678296 (Tsumura), WO94/18967 (Harvard),WO95/08327 (Dept. of Health and Human Services) (see also U.S. Pat. No.4,590,201 (Merck)) and EP-A-652000 (Eli Lilly).

[0027] Examples of peptidic angiogenesis inhibitors are described inWO94/02446 (British Biotech), WO94/02447 (British Biotech), WO94/21625(British Biotech), WO94/24140 (British Biotech), WO95/04033 (Celltech),EP-A-589719 (Eli Lilly), U.S. Pat. No. 5,399,667 (Frazier), EP-A-241830(The General Hospital Corporation) and WO97/38009 (Merck).

[0028] Particular angiogenesis inhibitors under development includethose listed in Table 3 below: TABLE 3 Angiogenesis Inhibitors CompoundTarget indications Company Commemts Tecogalan sodium Kaposi's sarcomaDaiichi sulfated poly- Solid tumors saccharide peptido- glycan complexAGM-1470 Kaposi's sarcoma Takeda/ Fumagillin analog Malignant tumorsAbbott CM101 Cancer Carbomed Polysaccharide Metastasis exotoxinMitoflaxone Solid tumors Lipha GM-1603 Glycomed Modified heparin rPF4Kaposi's sarcoma Replistatin Recombinant human Colon cancer Repligenplatelet factor-4 Glioma Renal cell carcinoma Malignant melanoma MPF-4Lilly Modified human platelet factor-4 Recombinant EntreMed angiostatinEndostatin collagen fragment Thalidomide Brain, breast and EntreMedprostate cancer DC101 ImClone Monoclonal Systems antibody OLX-514 Solidtumors Aronex Sepsis Raloxifene Lilly hydrochloride Suramin sodiumMetastatic Parke-Davis hormone- refractory prostate carcinoma IL-12Kidney cancer Roche Marimastat Pancreatic, lung British and brain cancerBiotech CAI Wide range of NCI Ca channel blocker cancers

[0029] as well as the following peptidic and non-peptidic drugcompounds:

[0030] Other known compounds capable of targeting regions ofangiogenesis are listed in Table 4 below: TABLE 4 Vector molecules withknown affinity for receptors associated with angiogenesis VectorMolecules angiopoietins angiostatin [plasminogen fragment] [inhibitor]angiotensin II α₂-antiplasmin combinatorial libraries, compounds fromfor instance compounds that bind to basement membrane after degradationβ-Cyclodextrin tetradecasulfate endoglin endosialin endostatin [collagenfragment] epidermal growth factor (heparin-binding) fibrinfibrinopeptide B fibroblast growth factor, FGF-3, basic fibronectinfumagillin and analogs heparin hepatocyte growth factor hyaluronaninsulin-like growth factor interferon-α, β [inhibitors] interleukins:IL-8, IL-12 [inhibitor] laminin, laminin fragments leukemia inhibitoryfactor linomide matrix metalloproteinase-2 metalloproteinasesmetalloproteinase inhibitors monoclonal antibodies for instance: toangiogenic factors or their receptors or to components of thefibrinolytic system peptides: for instance, cyclic RGD_(D)FV placentalgrowth factor placental proliferin-related protein plasminogenplasminogen activator plasminogen activator inhibitor-1 plateletactivating factor antagonists [inhibitors] platelet-derived growthfactor (e.g. type BB) platelet-derived growth factor receptorsplatelet-derived endothelial cell growth factor pleiotropin proliferin,proliferin-related protein selectins: E-selectin SPARC snake venoms(RGD-containing) substance P (a neuropeptide: neurokinin) suramin tissueinhibitor of metalloproteinases (e.g. TIMP-2) thalidomide thrombinthrombin-receptor-activating tetradecapeptide transforming growthfactor-α, β transforming growth factor receptor tumor growth factor-αtumor necrosis factor vitronectin

[0031] Similarly the compounds described in WO95/08327 may be used asvectors (see also Kohn et al. Proc. Nat. Acad Sci. USA 92: 1307-1311(1995) and J. Clin. Oncol. 15: 1985-1993 (1997)).

[0032] Particular examples of vector compounds described in some of thepatent publications mentioned above are as follows:

[0033] WO95/08327 (Dept. of Health and Human Services) describesangiogenesis inhibitor compounds of formula I and II:

Y—(CH₂)_(p)—Ar¹—X—Ar²  (I)

[0034] wherein

[0035] Ar¹ and Ar² are aromatic groups and may be different or the same;and X is a linking group eg. O, S, SO₂, CO, CHCN, alkyl, alkoxy oralkoxyalkyl, and

[0036] wherein

[0037] A is N or CH; R1 is H, —CONH, —CONHR⁵, COOH, —COOR⁵, SO₂NH₂; R2is H, NH₂, NHCOPh, —NHCOR⁵, —NHCHO, —NHR⁵, —N(R⁵)₂; and R⁵ is alkyl with1-6 carbons, e.g.

[0038] WO95/06473 (Nippon Kayaku Kabushiki Kasai) discloses antitumorand angiogenesis inhibitor compounds of formula 1 and 2:

[0039] wherein

[0040] X is O, COO; and M is a transition metal, and

[0041] wherein

[0042] X₁ and X₂ are substituted or unsubstituted phenyl or naphthylgroups; Y₁ and Y₂ are halogen atoms, amino groups or mono- ordi-substituted amino groups; and Z is NHC₂H₄NH or a substituted orunsubstituted aromatic diamine residue e.g.

[0043] WO95/04033 (Celltech Limited) discloses the followingangiogenesis inhibitors:

[0044] R3 is H, halogen or CH₃, CF₃ or OCH₃; R2 is H or CH₃ e.g.N′-hydroxy-Nl-(1-(S)-carbamoyl-2,2-dimethylpropyl)-2-(R)-4(chlorophenyl-propyl)succinamide;N⁴-hydroxy-N¹-(1-(S)-carbamoyl-2,2-dimethylpropyl)-2-(R)-(4-methylphenyl-propyl)succinamide;N⁴-hydroxy-N-(1-(S)-carbamoyl-2,2-dimethylpropyl)-2-(R)-(4-methoxyphenyl-propyl)succinamide;andN⁴-hydroxy-N¹-(1-(S)-carbamoyl-2,2-dimethylpropyl)-2-(R)-(4-trifluoromethylphenyl-propyl)-succinamide.

[0045] EP 241830 (The General Hospital Corporation) disclosespurification of hepatoma-derived growth factor (HDGF), which is anendothelial mitogen and a potent angiogenic factor. The use of HDGF incontrolling angiogenesis and detecting cancerous liver tumors by use ofan immunodiagnostic assay is also disclosed. The HDGF peptide fragmenthas an N-terminal amino acid sequence wherein the first 16 amino acidsare:

[0046] leu-pro-ala-leu-pro-glu-asp-gly-gly-xx-gly-ala-phe-pro-pro-gly

[0047] (xx=unidentified amino acid moiety)

[0048] An HDGF peptide fragment is also disclosed which has anN-terminal amino acid extension sequence comprising:

[0049](ala/ser)-(leu/arg)-pro-(ala/gly)-(leu/pro)-ala-gly-thr-met-ala-(ala)-gly-ser-(isoleu)-thr-thr-leu

[0050] EP 652000 (Eli Lilly and Company) discloses angiogenesisinhibitor and angiogenic disease inhibitor compounds having the forumla:

[0051] wherein

[0052] R1 and R3 are H, Me, —C(O) (C₁-C₆ alkyl), —C(O)Ar; Ar isoptionally substituted phenyl; and R2 is pyrrolidino or piperidino, e.g.

[0053] EP 652000 [589719?] (Eli Lilly and Company) discloses modifiedplatelet factor-4 having the amino acid sequence:

[0054] MPF-4

[0055]NH₂-Ser-Gln-Val-Arg-Pro-Arg-His-Ile-Thr-Ser-Leu-Glu-Val-Ile-Lys-Ala-Gly-Pro-His-Cys-Pro-Thr-Ala-Gln-Leu-Ile-Ala-Thr-Leu-Lys-Asn-Gly-Arg-Lys-Ile-Cys-Leu-Asp-Leu-Gln-Ala-Pro-Leu-Tyr-Lys-Lys-Ile-Ile-Lys-Lys-Leu-Leu-Glu-Ser-COOH

[0056] CPF-4

[0057]NH₂-Ser-Gln-Val-Arg-Pro-Arg-His-Ile-Thr-Ser-Leu-Glu-Val-Ile-Lys-Ala-Gly-Pro-His-Cys-Pro-Thr-Ala-Gln-Leu-Ile-Ala-Thr-Leu-Lys-Asn-Gly-Arg-Lys-Ile-Cys-Leu-Asp-Leu-Gln-Ala-Pro-Leu-Tyr-Lys-Ile-Ile-Lys-Lys-Leu-Leu-Glu-Ser-COOHdisulfide bonded to a second protein having

[0058] the amino acid sequenceNH₂-Glu-Ala-Glu-Glu-Asp-Gly-Asp-Leu-Gln-Cys-Leu-Cys-Val-Lys-Thr-Thr-COOH.There are disulfide bridges between Cys-20 of MPF-4 and Cys-10 of thesaid second protein and between Cys-36 of MPF-4 and Cys-12 of the saidsecond protein.

[0059] WO94/13277 (Imperical Cancer Research Technology Limited)discloses the use of compounds of formula I:

[0060] wherein

[0061] R1 to R4 are each independently one or more of —X, N₃, —NO₂,halo, trifluoromethyl, R⁵, OR⁵, —CH₂OR⁶, —COR⁵, —CH₂OCOR⁵, —NHCOR⁵,—CH₂NHCOR⁵, —NR⁵R⁶, —CH₂NR⁵R⁶, —CH₂NO₂, CONR⁵R⁶, CH₂CONR⁵R⁶, —COOR⁵,—CH₂COOR⁵, —CHO and CH₂CHO and —X is independently —SO₃R⁵, —CH₂PO₃R⁵R⁶,—CH₂SO₃R⁵, OSO₃R⁵, —CH₂OSO₃R⁵, —NHSO₃R⁵, —CH₂NHSO₃R⁵, OPO₃R⁵R⁶,—CH₂OPO₃R⁵R⁶ and —PO₃R⁵R⁶ where R⁵ and R⁶ are chosen independently from—H and lower alkyl and wherein A is a chemical group comprising at least5 and no more than 30 bonds directly linking the naphthyl groupsprovided that (i) the compound is not suramin and (ii) when A is not

[0062] wherein

[0063] m and n are independently 0, 1 or 2, then at least one of R1 toR4 is —OH or an acidic group; and of the pharmaceutically acceptablesalts, esters, salts of such esters or amides of such compounds.

[0064] Also described are compounds wherein the linkage of A to thenaphthyl ring is via an amide or sulphonamide group. Furthermore A mayin some cases be a group of formula II. A may also be selected fromstraight chain or branched alkyl groups, aryl groups, alkylaryl groups,aliphatic dicarboxylic acids, polyenes and derivatives thereof andpolyols and derivatives thereof. Some further compounds are of formulae:

[0065] EP 678296 (Tsumura & Co.) discloses angiogenesis inhibitors ofthe general formula:

[0066] WO94/18967 (President and Fellows of Harvard College) discloses aclass of imidazoles that inhibit angiogenesis:

[0067] EP-A-618208 (Daiichi Pharmaceutical) discloses compounds offormula:

[0068] WO94/02446 (British Biotechnology Limited) discloses compounds offormula:

[0069] WO94/02447 (British Biotechnology Limited) discloses compounds offormula:

[0070] WO94/21625 (British Biotechnology Limited) discloses compounds offormula:

[0071] WO94/24149 (British Biotechnology Limited) discloses compounds offormula (I):

[0072] wherein

[0073] X is —CONHOH or COOH, principally characterised by the presencein substituent R3 and/or R4 of a group of formula (II)

[0074] The contents of all the publications referred to herein is herebyincorporated by reference.

[0075] Particularly preferred vectors include amino acid derivativessuch as described in WO94/02446, hydroxamic acid derivatives such asdescribed in WO94/02447, thiazolopyrimidines such as described inEP-A-618208, triazoles such as described in WO95/08327, quinazolinessuch as described in WO97/30035, isoindolones such as described inWO97/37655, integrin inhibitors, VEGF antagonists, bFGF antagonists,thrombospondin and thrombospondin fragments, CD36 and growth factors(e.g. VEGF, bFGF, etc).

[0076] CAM-D and other candidate identification and evaluationtechniques as mentioned above can also be used to find or assess furthercandidate peptidic and non-peptidic vectors.

[0077] Thus it is also possible to obtain molecules that bindspecifically to angiogenesis associated receptors by direct screening ofmolecular libraries. Screening of peptidic libraries may also be used toidentify generally effective peptidic structures of which non-peptidicanalogs may be generated by conventional or combinatorial chemistry.Binding moieties identified in this way may be coupled to a linkermolecule, constituting a general tool for attaching any vector molecule(or molecules) to the reporter. Vector molecules may be generated fromcombinatorial libraries without necessarily knowing the exact moleculartarget, by functionally selecting (in vitro, ex vivo or in vivo) formolecules binding to the region/structure to be imaged.

[0078] As mentioned above, the agents of formula I comprise vector,linker and reporter moieties. A linker moiety may serve to link onevector to one reporter; alternatively it may link together more thanvector and/or more than one reporter. Likewise a reporter or a vectormay be linked to more than one linker. Use in this way of a plurality ofreporters (eg. several linker-reporter moieties attached to one vectoror several reporters attached to one linker itself attached to onevector) may enable the detectability of the contrast agent to beincreased (eg. by increasing its radioopacity, echogenicity orrelaxivity) or may enable it to be detected in more than one imagingmodality. Use in this way of a plurality of vectors may increase thetargeting efficiency of the contrast agent or may make the contrastagent able to target more than one site, eg. different receptors for anagent which has receptor heterogeneity. Thus for example the agent offormula I may include vector moieties with affinity sites other thanangiogenesis associated receptors, eg. with affinities for cell surfaceson body duct wall surfaces. Accordingly, the agent may include vectorssuch as antibody fragments and oligopeptides, eg. containing RGD oranalogous cell surface binding peptide motifs (for example as describedin EP-A-422937 and EP-A-422938 (Merck)) or other vectors as described inGB 9700699.3. Such extra vectors may also be selected from any of themolecules that naturally concentrate in a selected target organ, tissue,cell or group of cells, or other location in a mammalian body, in vivo.These can include amino acids, oligopeptides (e.g. hexapeptides),molecular recognition units (MRU's), single chain antibodies (SCA's),proteins, non-peptide organic molecules, Fab fragments, and antibodies.Examples of site-directed molecules include polysaccharides (e.g. CCKand hexapeptides), proteins (such as lectins, asialofetuin, polyclonalIgG, blood clotting proteins (e.g. hirudin), lipoproteins andglycoproteins), hormones, growth factors, clotting factors (such asPF4), polymerized fibrin fragments (e.g., E₁), serum amyloid precursor(SAP) proteins, low density lipoprotein (LDL) precursors, serum albumin,surface proteins of intact red blood cells, receptor binding moleculessuch as estrogens, liver-specific proteins/polymers such asgalactosyl-neoglycoalbumin (NGA) (see Vera et al. in Radiology 151: 191(1984)) N-(2-hydroxy-propyl)methacrylamide (HMPA) copolymers withvarying numbers of bound galactosamines (see Duncan et al., Biochim.Biophys. Acta 880:62 (1986)), and allyl and 6-aminohexyl glycosides (seeWong et al., Carbo. Res. 170:27 (1987)), and fibrinogen. Thesite-directed protein can also be an antibody. The choice of antibody,particularly the antigen specificity of the antibody, will depend on theparticular intended target site for the agent. Monoclonal antibodies arepreferred over polyclonal antibodies. Preparation of antibodies thatreact with a desired antigen is well known. Antibody preparations areavailable commercially from a variety of sources. Fibrin fragment E1 canbe prepared as described by Olexa et al. in J. Biol. Chem. 254:4925(1979). Preparation of LDL precursors and SAP proteins is described byde Beer et al. in J. Immunol. Methods 50:17 (1982). The above describedarticles are incorporated herein by reference in their entirety.

[0079] It is especially preferred that such extra vectors should bind soas to slow but not prevent the motion of the agent in the bloodstreamand to anchor it in place when it is bound to a receptor site associatedwith angiogenesis.

[0080] Functional groups (e.g. amino groups, hydroxyl groups, carboxygroups, thiol groups, etc) on the vector compound may be used forbinding of the vector to the linker moiety or directly to the reportermoiety, e.g. using conventional chemical coupling techniques.

[0081] Where the vector is a peptidic compound, the reporter is amultireporter, e.g. a metallated polychelant (preferably a dendrimericpolychelant), a magnetic (preferably superparamagnetic) particle, avesicle containing contrast effective particles or a solution ofcontrast effective molecule, a polyionic species (e.g. a polymercarrying a multiplicity of ionic groups, preferably anionic groups, e.g.a carboxylate, phosphate or sulphonate polymer).

[0082] Where the vector is non-peptidic, the reporter may be amultireporter or alternatively may comprise one or a small number (e.g.up to 10) of detectable labels, e.g. chelated paramagnetic metal ions,covalently bound or chelated radioisotopes, and chromophores (orfluorophores, etc). Where the reporter is or comprises a covalentlybound radionuclide, this is preferably an iodine radionuclide ratherthan a tritium or ¹³C atom

[0083] Linker

[0084] A wide variety of linkers can be used, including biodegradablelinkers and biopolymers.

[0085] The linker component of the contrast agent is at its simplest abond between the vector and reporter moieties. More generally howeverthe linker will provide a mono- or multi-molecular skeleton covalentlyor non-covalently linking one or more vectors to one or more reporters,eq. a linear, cyclic, branched or reticulate molecular skeleton, or amolecular aggregate, with in-built or pendant groups which bindcovalently or non-covalently, eq. coordinatively, with the vector andreporter moieties or which encapsulate, entrap or anchor such moieties.

[0086] Thus linking of a reporter unit to a desired vector may beachieved by covalent or non-covalent means, usually involvinginteraction with one or more functional groups located on the reporterand/or vector. Examples of chemically reactive functional groups whichmay be employed for this purpose include amino, hydroxyl, sulfhydryl,carboxyl, and carbonyl groups, as well as carbohydrate groups, vicinaldiols, thioethers, 2-aminoalcohols, 2-aminothiols, guanidinyl,imidazolyl and phenolic groups.

[0087] Covalent coupling of reporter and vector may therefore beeffected using linking agents containing reactive moities capable ofreaction with such functional groups. Examples of reactive moietiescapable of reaction with sulfhydryl groups include a-haloacetylcompounds of the type X—CH₂CO— (where X=Br, Cl or I), which showparticular reactivity for sulfhydryl groups but which can also be usedto modify imidazolyl, thioether, phenol and amino groups as described byGurd, F.R.N. in Methods Enzymol. (1967) 11, 532. N-Maleimide derivativesare also considered selective towards sulfhydryl groups, but mayadditionaly be useful in coupling to amino groups under certainconditions. Reagents such as 2-iminothiolane, e.g. as described byTraut, R. et al. in Biochemistry (1973) 12, 3266, which introduce athiol group through conversion of an amino group, may be considered assulfhydryl reagents if linking occurs through the formation ofdisulphide bridges. Thus reagents which introduce reactive disulphidebonds into either the reporter or the vector may be useful, sincelinking may be brought about by disulphide exchange between the vectorand reporter; examples of such reagents include Ellman's reagent (DTNB),4,4′-dithiodipyridine, methyl-3-nitro-2-pyridyl disulphide andmethyl-2-pyridyl disulphide (described by Kimura, T. et al. in Analyt.Biochem. (1982) 122, 271).

[0088] Examples of reactive moieties capable of reaction with aminogroups include alkylating and acylating agents. Representativealkylating agents include:

[0089] i) α-haloacetyl compounds, which show specificity towards aminogroups in the absence of reactive thiol groups and are of the typeX—CH₂CO— (where X=Cl, Br or I), e.g. as described by Wong, Y-H.H. inBiochemistry (1979) 24, 5337;

[0090] ii) N-maleimide derivatives, which may react with amino groupseither through a Michael type reaction or through acylation by additionto the ring carbonyl group as described by Smyth, D. G. et al. in J. Am.Chem. Soc. (1960) 82, 4600 and Biochem. J. (1964) 91, 589;

[0091] iii) aryl halides such as reactive nitrohaloaromatic compounds;

[0092] iv) alkyl halides as described by McKenzie, J. A. et al. in J.Protein Chem. (1988) 7, 581;

[0093] v) aldehydes and ketones capable of Schiff's base formation withamino groups, the adducts formed usually being stabilised throughreduction to give a stable amine;

[0094] vi) epoxide derivatives such as epichlorohydrin andbisoxiranes,which may react with amino, sulfhydryl or phenolic hydroxylgroups;

[0095] vii) chlorine-containing derivatives of s-triazines, which arevery reactive towards nucleophiles such as amino, sufhydryl and hydroxygroups;

[0096] viii) aziridines based on s-triazine compounds dET_(A)iled above,e.g. as described by Ross, W.C.J. in Adv. Cancer Res. (1954) 2, 1, whichreact with nucleophiles such as amino groups by ring opening;

[0097] ix) squaric acid diethyl esters as described by Tietze, L. F. inChem. Ber. (1991) 124, 1215; and

[0098] x) α-haloalkyl ethers, which are more reactive alkylating agentsthan normal alkyl halides because of the activation caused by the etheroxygen atom, e.g. as described by Benneche, T. et al. in Eur. J. Med.Chem. (1993) 28, 463.

[0099] Representative amino-reactive acylating agents include:

[0100] i) isocyanates and isothiocyanates, particularly aromaticderivatives, which form stable urea and thiourea derivativesrespectively and have been used for protein crosslinking as described bySchick, A. F. et al. in J. Biol. Chem. (1961) 236, 2477;

[0101] ii) sulfonyl chlorides, which have been described by Herzig, D.J. et al. in Biopolymers (1964) 2, 349 and which may be useful for theintroduction of a fluorescent reporter group into the linker;

[0102] iii) Acid halides;

[0103] iv) Active esters such as nitrophenylesters orN-hydroxysuccinimidyl esters;

[0104] v) acid anhydrides such as mixed, symmetrical orN-carboxyanhydrides;

[0105] vi) other useful reagents for amide bond formation as describedby Bodansky, M. et al. in ‘Principles of Peptide Synthesis’ (1984)Springer-Verlag;

[0106] vii) acylazides, e.g. wherein the azide group is generated from apreformed hydrazide derivative using sodium nitrite, e.g. as describedby Wetz, K. et al. in Anal. Biochem. (1974) 58, 347;

[0107] viii) azlactones attached to polymers such as bis-acrylamide,e.g. as described by Rasmussen, J. K. in Reactive Polymers (1991) 16,199; and

[0108] ix) Imidoesters , which form stable amidines on reaction withamino groups, e.g. as described by Hunter, M. J. and Ludwig, M. L. in J.Am. Chem. Soc. (1962) 84, 3491.

[0109] Carbonyl groups such as aldehyde functions may be reacted withweak protein bases at a pH such that nucleophilic protein side-chainfunctions are protonated. Weak bases include 1,2-aminothiols such asthose found in N-terminal cysteine residues, which selectively formstable 5-membered thiazolidine rings with aldehyde groups, e.g. asdescribed by Ratner, S. et al. in J. Am. Chem. Soc. (1937) 59, 200.Other weak bases such as phenyl hydrazones may be used, e.g. asdescribed by Heitzman, H. et al. in Proc. Natl. Acad. Sci. USA (1974)71, 3537.

[0110] Aldehydes and ketones may also be reacted with amines to formSchiff's bases, which may advantageously be stabilised through reductiveamination. Alkoxylamino moieties readily react with ketones andaldehydes to produce stable alkoxamines, e.g. as described by Webb, R.et al. in Bioconjugate Chem. (1990) 1, 96.

[0111] Examples of reactive moieties capable of reaction with carboxylgroups include diazo compounds such as diazoacetate esters anddiazoacetamides, which react with high specificity to generate estergroups, e.g. as described by Herriot R. M. in Adv. Protein Chem. (1947)3, 169. Carboxylic acid modifying reagents such as carbodiimides, whichreact through O-acylurea formation followed by amide bond formation, mayalso usefully be employed; linking may be facilitated through additionof an amine or may result in direct vector-receptor coupling. Usefulwater soluble carbodiimides include1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimido (CMC) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), e.g. as describedby Zot, H. G. and Puett, D. in J. Biol. Chem. (1989) 264, 15552. Otheruseful carboxylic acid modifying reagents include isoxazoliumderivatives such as Woodwards reagent K; chloroformates such asp-nitrophenylchloroformate; carbonyldiimidazoles such as1,1′-carbonyldiimidazole; and N-carbalkoxydihydroquinolines such asN-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline.

[0112] Other potentially useful reactive moieties include vicinal dionessuch as p-phenylenediglyoxal, which may be used to react with guanidinylgroups, e.g. as described by Wagner et al. in Nucleic acid Res. (1978)5, 4065; and diazonium salts, which may undergo electrophilicsubstitution reactions, e.g. as described by Ishizaka, K. and IshizakaT. in J. Immunol. (1960) 85, 163. Bis-diazonium compounds are readilyprepared by treatment of aryl diamines with sodium nitrite in acidicsolutions. It will be appreciated that functional groups in the reporterand/or vector may if desired be converted to other functional groupsprior to reaction, e.g. to confer additional reactivity or selectivity.Examples of methods useful for this purpose include conversion of aminesto carboxylic acids using reagents such as dicarboxylic anhydrides;conversion of amines to thiols using reagents such asN-acetylhomocysteine thiolactone, S-acetylmercaptosuccinic anhydride,2-iminothiolane or thiol-containing succinimidyl derivatives; conversionof thiols to carboxylic acids using reagents such as α-haloacetates;conversion of thiols to amines using reagents such as ethylenimine or2-bromoethylamine; conversion of carboxylic acids to amines usingreagents such as carbodiimides followed by diamines; and conversion ofalcohols to thiols using reagents such as tosyl chloride followed bytransesterification with thioacetate and hydrolysis to the thiol withsodium acetate.

[0113] Vector-reporter coupling may also be effected using enzymes aszero-length crosslinking agents; thus, for example, transglutaminase,peroxidase and xanthine oxidase have been used to produce crosslinkedproducts. Reverse proteolysis may also be used for crosslinking throughamide bond formation.

[0114] Non-covalent vector-reporter coupling may, for example, beeffected by electrostatic charge interactions, through chelation in theform of stable metal complexes or through high affinity bindinginteraction.

[0115] A vector which is coupled to a peptide, lipo-oligosaccharide orlipopeptide linker which contains a element capable of mediatingmembrane insertion may also be useful. One example is described byLeenhouts, J. M. et al. in Febs Letters (1995) 370(3), 189-192.

[0116] Coupling may also be effected using avidin or streptavidin, whichhave four high affinity binding sites for biotin. Avidin may thereforebe used to conjugate vector to reporter if both vector and reporter arebiotinylated. Examples are described by Bayer, E. A. and Wilchek, M. inMethods Biochem. Anal. (1980) 26, 1. This method may also be extended toinclude linking of reporter to reporter, a process which may encourageassociation of the agent and consequent potentially increased efficacy.Alternatively, avidin or streptavidin may be attached directly to thesurface of reporter particles.

[0117] Non-covalent coupling may also utilise the bifunctional nature ofbispecific immunoglobulins. These molecules can specifically bind twoantigens, thus linking them. For example, either bispecific IgG orchemically engineered bispecific F(ab)′₂ fragments may be used aslinking agents. Heterobifunctional bispecific antibodies have also beenreported for linking two different antigens, e.g. as described by Bode,C. et al. in J. Biol. Chem. (1989) 264, 944 and by Staerz, U. D. et al.in Proc. Natl. Acad. Sci. USA (1986) 83, 1453. Similarly, any reporterand/or vector containing two or more antigenic determinants (e.g. asdescribed by Chen, Aa et al. in Am. J. Pathol. (1988) 130, 216) may becrosslinked by antibody molecules and lead to formation of cross-linkedassemblies of agents of formula I of potentially increased efficacy.

[0118] So-called zero-length linking agents, which induce directcovalent joining of two reactive chemical groups without introducingadditional linking material (e.g. as in amide bond formation inducedusing carbodiimides or enzymatically) may, if desired, be used inaccordance with the invention, as may agents such as biotin/avidinsystems which induce non-covalent reporter-vector linking and agentswhich induce electrostatic interactions.

[0119] Most commonly, however, the linking agent will comprise two ormore reactive moieties, e.g. as described above, connected by a spacerelement. The presence of such a spacer permits bifunctional linkers toreact with specific functional groups within a molecule or between twodifferent molecules, resulting in a bond between these two componentsand introducing extrinsic linker-derived material into thereporter-vector conjugate. The reactive moieties in a linking agent maybe the same (homobifunctional agents) or different (heterobifunctionalagents or, where several dissimilar reactive moieties are present,heteromultifunctional agents), providing a diversity of potentialreagents that may bring about covalent bonding between any chemicalspecies, either intramolecularly or intermolecularly.

[0120] The nature of extrinsic material introduced by the linking agentmay have a critical bearing on the targeting ability and generalstability of the ultimate product. Thus it may be desirable to introducelabile linkages, e.g. containing spacer arms which are biodegradable orchemically sensitive or which incorporate enzymatic cleavage sites.Alternatively the spacer may include polymeric components, e.g. to actas surfactants and enhance the stability of the agent. The spacer mayalso contain reactive moieties, e.g. as described above to enhancesurface crosslinking.

[0121] Spacer elements may typically consist of aliphatic chains whicheffectively separate the reactive moieties of the linker by distances ofbetween 5 and 30 Å. They may also comprise macromolecular structuressuch as poly(ethylene glycols). Such polymeric structures, hereinafterreferred to as PEGs, are simple, neutral polyethers which have beengiven much attention in biotechnical and biomedical applications (seee.g. Milton Harris, J. (ed) “Poly(ethylene glycol) chemistry,biotechnical and biomedical applications” Plenum Press, New York, 1992).PEGs are soluble in most solvents, including water, and are highlyhydrated in aqueous environments, with two or three water moleculesbound to each ethylene glycol segment; this has the effect of preventingadsorption either of other polymers or of proteins onto PEG-modifiedsurfaces. PEGs are known to be nontoxic and not to harm active-proteinsor cells, whilst covalently linked PEGs are known to be non-immunogenicand non-antigenic. Furthermore, PEGs may readily be modified and boundto other molecules with only little effect on their chemistry. Theiradvantageous solubility and biological properties are apparent from themany possible uses of PEGs and copolymers thereof, including blockcopolymers such as PEG-polyurethanes and PEG-polypropylenes.

[0122] Appropriate molecular weights for PEG spacers used in accordancewith the invention may, for example, be between 120 Daltons and 20kDaltons.

[0123] The major mechanism for uptake of particles by the cells of thereticuloendothelial system (RES) is opsonisation by plasma proteins inblood; these mark foreign particles which are then taken up by the RES.The biological properties of PEG spacer elements used in accordance withthe invention may serve to increase the circulation time of the agent ina similar manner to that observed for PEGylated liposomes (see e.g.Klibanov, A. L. et al. in FEBS Letters (1990) 268, 235-237 and Blume, G.and Cevc, G. in Biochim. Biophys. Acta (1990) 1029, 91-97). Increasedcoupling efficiency to areas of interest may also be achieved usingantibodies bound to the terminii of PEG spacers (see e.g. Maruyama, K.et al. in Biochim. Biophys. Acta (1995) 1234, 74-80 and Hansen, C. B. etal. in Biochim. Biophys. Acta (1995) 1239, 133-144).

[0124] Other representative spacer elements include structural-typepolysaccharides such as polygalacturonic acid, glycosaminoglycans,heparinoids, cellulose and marine polysaccharides such as alginates,chitosans and carrageenans; storage-type polysaccharides such as starch,glycogen, dextran and aminodextrans; polyamino acids and methyl andethyl esters thereof, as in homo- and co-polymers of lysine, glutamicacid and aspartic acid; and polypeptides, oligosaccharides andoligonucleotides, which may or may not contain enzyme cleavage sites.

[0125] In general, spacer elements may contain cleavable groups such asvicinal glycol, azo, sulfone, ester, thioester or disulphide groups.Spacers containing biodegradable methylene diester or diamide groups offormula

—(Z)_(m).Y.X.C(R¹R²).X.Y.(Z)_(n)—

[0126] [where X and Z are selected from —O—, —S—, and —NR— (where R ishydrogen or an organic group); each Y is a carbonyl, thiocarbonyl,sulphonyl, phosphoryl or similar acid-forming group: m and n are eachzero or 1; and R¹ and R² are each hydrogen, an organic group or a group

[0127] —X.Y.(Z)_(m)—, or together form a divalent organic group] mayalso be useful; as discussed in, for example, WO-A-9217436 such groupsare readily biodegraded in the presence of esterases, e.g. in vivo, butare stable in the absence of such enzymes. They may thereforeadvantageously be linked to therapeutic agents to permit slow releasethereof.

[0128] Poly[N-(2-hydroxyethyl)methacrylamides] are potentially usefulspacer materials by virtue of their low degree of interaction with cellsand tissues (see e.g. Volfova, I., Rihova, B. and V. R. and Vetvicka, P.in J. Bioact. Comp. Polymers (1992) 7, 175-190). Work on a similarpolymer consisting mainly of the closely related 2-hydroxypropylderivative showed that it was endocytosed by the mononuclear phagocytesystem only to a rather low extent (see Goddard, P., Williamson, I.,Bron, J., Hutchkinson, L. E., Nicholls, J. and Petrak, K. in J. Bioct.Compat. Polym. (1991) 6, 4-24.).

[0129] Other potentially useful poymeric spacer materials include:

[0130] i) copolymers of methyl methacrylate with methacrylic acid; thesemay be erodible (see Lee, P. I. in Pharm. Res. (1993) 10, 980) and thecarboxylate substituents may cause a higher degree of swelling than withneutral polymers;

[0131] ii) block copolymers of polymethacrylates with biodegradablepolyesters (see e.g. San Roman, J. and Guillen-Garcia, P. inBiomaterials (1991) 12, 236-241);

[0132] iii) cyanoacrylates, i.e. polymers of esters of 2-cyanoacrylicacid—these are biodegradable and have been used in the form ofnanoparticles for selective drug delivery (see Forestier, F., Gerrier,P., Chaumard, C., Quero, A. M., Couvreur, P. and Labarre, C. in J.Antimicrob. Chemoter. (1992) 30, 173-179);

[0133] iv) polyvinyl alcohols, which are water-soluble and generallyregarded as biocompatible (see e.g. Langer, R. in J. Control. Release(1991) 16, 53-60);

[0134] v) copolymers of vinyl methyl ether with maleic anhydride, whichhave been stated to be bioerodible (see Finne, U., Hannus, M. and Urtti,A. in Int. J. Pharm. (1992) 78. 237-241);

[0135] vi) polyvinylpyrrolidones, e.g. with molecular weight less thanabout 25,000, which are rapidly filtered by the kidneys (see Hespe, W.,Meier, A. M. and Blankwater, Y. M. in Arzeim.-Forsch./Drug Res. (1977)27, 1158-1162);

[0136] vii) polymers and copolymers of short-chain aliphatichydroxyacids such as glycolic, lactic, butyric, valeric and caproicacids (see e.g. Carli, F. in Chim. Ind. (Milan) (1993) 75, 494-9),including copolymers which incorporate aromatic hydroxyacids in order toincrease their degradation rate (see Imasaki, K., Yoshida, M., Fukuzaki,H., Asano, M., Kumakura, M., Mashimo, T., Yamanaka, H. and Nagai. T. inInt. J. Pharm. (1992) 81, 31-38);

[0137] viii) polyesters consisting of alternating units of ethyleneglycol and terephthalic acid, e.g. Dacron^(R), which are non-degradablebut highly biocompatible;

[0138] ix) block copolymers comprising biodegradable segments ofaliphatic hydroxyacid polymers (see e.g. Younes, H., Nataf, P. R., Cohn,D., Appelbaum, Y. J., Pizov, G. and Uretzky, G. in Biomater. Artif.Cells Artif. Organs (1988) 16, 705-719), for instance in conjunctionwith polyurethanes (see Kobayashi, H., Hyon, S. H. and Ikada, Y. in“Water-curable and biodegradable prepolymers”J. Biomed. Mater. Res.(1991) 25, 1481-1494);

[0139] x) polyurethanes, which are known to be well-tolerated inimplants, and which may be combined with flexible “soft” segments, e.g.comprising poly(tetra methylene glycol), poly(propylene glycol) orpoly(ethylene glycol)) and aromatic “hard” segments, e.g. comprising4,4′-methylenebis(phenylene isocyanate) (see e.g. Ratner, B. D.,Johnston, A. B. and Lenk, T. J. in J. Biomed. Mater. Res: AppliedBiomaterials (1987) 21, 59-90; Sa Da Costa, V. et al. in J. Coll.Interface Sci. (1981) 80, 445-452 and Affrossman, S. et al. in ClinicalMaterials (1991) 8, 25-31);

[0140] xi) poly(1,4-dioxan-2-ones), which may be regarded asbiodegradable esters in view of their hydrolysable ester linkages (seee.g. Song, C. X., Cui, X. M. and Schindler, A. in Med. Biol. Eng.Comput. (1993) 31, S147-150), and which may include glycolide units toimprove their absorbability (see Bezwada, R. S., Shalaby, S. W. andNewman, H. D. J. in Agricultural and synthetic polymers:Biodegradability and utilization (1990) (ed Glass, J. E. and Swift, G.),167-174—ACS symposium Series, #433, Washington D.C., U.S.A.—AmericanChemical Society);

[0141] xii) polyanhydrides such as copolymers of sebacic acid(octanedioic acid) with bis(4-carboxy-phenoxy)propane, which have beenshown in rabbit studies (see Brem, H., Kader, A., Epstein, J. I.,Tamargo, R.J., Domb, A., Langer, R. and Leong, K. W. in Sel. CancerTher. (1989) 5, 55-65) and rat studies (see Tamargo, R. J., Epstein, J.I., Reinhard, C. S., Chasin, M. and Brem, H. in J. Biomed. Mater. Res.(1989) 23, 253-266) to be useful for controlled release of drugs in thebrain without evident toxic effects;

[0142] xiii) biodegradable polymers containing ortho-ester groups, whichhave been employed for controlled release in vivo (see Maa, Y. F. andHeller, J. in J. Control. Release (1990) 14, 21-28); and

[0143] xiv) polyphosphazenes, which are inorganic polymers consisting ofalternate phosphorus and nitrogen atoms (see Crommen, J. H., Vandorpe,J. and Schacht, E. H. in J. Control. Release (1993) 24, 167-180).

[0144] The following tables list linking agents which may be useful intargetable agents in accordance with the invention.

[0145] Heterobifunctional Linking Agents Linking agent Reactivity 1Reactivity 2 Comments ABH carbohydrate photoreactive ANB-NOS —NH₂photoreactive APDP (1) —SH photoreactive iodinable disulphide linker APG—NH₂ photoreactive reacts selectively with Arg at pH 7-8 ASIB (1) —SHphotoreactive iodinable ASBA (1) —COOH photoreactive iodinable EDC —NH₂—COOH zero-length linker GMBS —NH₂ —SH sulfo-GMBS —NH₂ —SH water-solubleHSAB —NH₂ photoreactive sulfo-HSAB —NH₂ photoreactive water-soluble MBS—NH₂ —SH sulfo-MBS —NH₂ —SH water-soluble M₂C₂H carbohydrate —SH MPBHcarbohydrate —SH NHS-ASA (1) —NH₂ photoreactive iodinable sulfo-NHS-—NH₂ photoreactive water-soluble, ASA (1) iodinable sulfo-NHS-LC- —NH₂photoreactive water-soluble, ASA (1) iodinable PDPH carbohydrate —SHdisulphide linker PNP-DTP —NH₂ photoreactive SADP —NH₂ photoreactivedisulphide linker sulfo-SADP —NH₂ photoreactive water-soluble disulphidelinker SAED —NH₂ photoreactive disulphide linker SAND —NH₂ photoreactivewater-soluble disulphide linker SANPAH —NH₂ photoreactive sulfo-SANPAH—NH₂ photoreactive water-soluble SASD (1) —NH₂ photoreactivewater-soluble iodinable disulphide linker SIAB —NH₂ —SH sulfa-SIAB —NH₂—SH water-soluble SMCC —NH₂ —SH sulfo-SMCC —NH₂ —SH water-soluble SMPB—NH₂ —SH sulfo-SMPB —NH₂ —SH water-soluble SMPT —NH₂ —SH sulfo-LC-SMPT—NH₂ —SH water-soluble SPDP —NH₂ —SH sulfo-SPDP —NH₂ —SH water-solublesulfo-LC-SPDP —NH₂ —SH water-soluble sulfo-SAMCA (2) —NH₂ photoreactivesulfo-SAPB —NH₂ photoreactive water-soluble

[0146] Homobifunctional Linking Agents Linking agent Reactivity CommentsBS —NH₂ BMH —SH BASED (1) photoreactive iodinable disulphide linkerBSCOES —NH₂ sulfo-BSCOES —NH₂ water-soluble DFDNB —NH₂ DMA —NH₂ DMP —NH₂DMS —NH₂ DPDPB —SH disulphide linker DSG —NH₂ DSP —NH₂ disulphide linkerDSS —NH₂ DST —NH₂ sulfo-DST —NH₂ water-soluble DTBP —NH₂ disulphidelinker DTSSP —NH₂ disulphide linker EGS —NH₂ sulfo-EGS —NH₂water-soluble SPBP —NH₂

[0147] Biotinylation Agents Agent Reactivity Comments biotin-BMCC —SHbiotin-DPPE* preparation of biotinylated liposomes biotin-LC-DPPE*preparation of biotinylated liposomes biotin-HPDP —SH disulphide linkerbiotin-hydrazide carbohydrate biotin-LC-hydrazide carbohydrateiodoacetyl-LC-biotin —NH₂ NHS-iminobiotin —NH₂ reduced affinity foravidin NHS-SS-biotin —NH₂ disulphide linker photoactivatable biotinnucleic acids sulfo-NHS-biotin —NH₂ water-soluble sulfo-NHS-LC-biotin—NH₂

[0148] Agents for Protein Modification Agent Reactivity FunctionEllman's reagent —SH quantifies/detects/protects DTT —S.S— reduction2-mercaptoethanol —S.S— reduction 2-mercaptylamine —S.S— reductionTraut's reagent —NH₂ introduces —SH SATA —NH₂ introduces protected —SHAMCA-NHS —NH₂ fluorescent labelling AMCA-hydrazide carbohydratefluorescent labelling AMCA-HPDP —S.S— fluorescent labelling SBF-chloride—S.S— fluorescent detection of —SH N-ethylmaleimide —S.S— blocks —SHNHS-acetate —NH₂ blocks and acetylates —NH₂ citraconic —NH₂ reversiblyblocks and anhydride introduces negative charges DTPA —NH₂ introduceschelator BNPS-skatole tryptophan cleaves tryptophan residueBolton-Hunter —NH₂ introduces iodinable group para-iodophenylalanine

[0149] In addition to the already contemplated straight chain andbranched PEG-like linkers (e.g polyethylene glycols and other containing2 to 100 recurring units of ethylene oxide), linkers in the VLR systemcan be independently a chemical bond or the residue of a linking group.The phrase “residue of a linking group” as used herein refers to amoiety that remains, results, or is derived from the reaction of avector reactive group with a reactive site on a vector. The phrase“vector reactive group” as used herein refers to any group which canreact with functional groups typically found on vectors, thederivatization of which only minimally effects the ability of the vectorto bind to its receptor. However, it is specifically contemplated thatsuch vector reactive groups can also react with functional groupstypically found on relevant protein molecules. Thus, in one aspect thelinkers useful in the practice of this invention derive from thosegroups which can react with any relevant molecule which comprises avector as described above containing a reactive group, whether or notsuch relevant molecule is a protein, to form a linking. group.

[0150] Preferred linking groups are derived from vector reactive groupsselected from but not limited to:—

[0151] a group that will react directly with carboxy, aldehyde, amine(NHR), alcohols, sulfhydryl groups, activated methylenes and the like,on the vector, for example, active halogen containing groups including,for example, chloromethylphenyl groups and chloroacetyl [ClCH₂C(═O)—]groups, activated 2-(leaving group substituted)-ethylsulfonyl andethylcarbonyl groups such as 2-chloroethylsulfonyl and2-chloroethylcarbonyl; vinylsulfonyl; vinylcarbonyl; epoxy; isocyanato;isothiocyanato; aldehyde; aziridine; succinimidoxycarbonyl; activatedacyl groups such as carboxylic acid halides; mixed anhydrides and thelike.

[0152] A group that can react readily with modified vector moleculescontaining a vector reactive group, i.e., vectors containing a reactivegroup modified to contain reactive groups such as those mentioned in thetables above, for example, by oxidation of the vector to an aldehyde ora carboxylic acid, in which case the “linking group” can be derived fromreactive groups selected from amino, alkylamino, arylamino, hydrazino,alkylhydrazino, arylhydrazino, carbazido, semicarbazido, thiocarbazido,thiosemicarbazido, sulfhydryl, sulfhydrylalkyl, sulfhydrylaryl, hydroxy,carboxy, carboxyalkyl and carboxyaryl. The alkyl portions of saidlinking groups can contain from 1 to about 20 carbon atoms. The arylportions of said linking groups can contain from about 6 to about 20carbon atoms; and

[0153] a group that can be linked to the vector containing a reactivegroup, or to the modified vector as noted above by use of a crosslinkingagent. The residues of certain useful crosslinking agents, such as, forexample, homobifunctional and heterobifunctional gelatin hardeners,bisepoxides, and bisisocyanates can become a part of a linking groupduring the crosslinking reaction. Other useful crosslinking agents,however, can facilitate the crosslinking, for example, as consumablecatalysts, and are not present in the final conjugate. Examples of suchcrosslinking agents are carbodiimide and carbamoylonium crosslinkingagents as disclosed in U.S. Pat. No. 4,421,847 and the ethers of U.S.Pat. No. 4,877,724. With these crosslinking agents, one of the reactantssuch as the vector must have a carboxyl group and the other such as along chain spacer must have a reactive amine, alcohol, or sulfhydrylgroup. In amide bond formation, the crosslinking agent first reactsselectively with the carboxyl group, then is split out during reactionof the thus “activated” carboxyl group with an amine to form an amidelinkage between thus covalently bonding the two moieties. An advantageof this approach is that crosslinking of like molecules, e.g., vector tovector is avoided, whereas the reaction of, for example,homo-bifunctional crosslinking agents is nonselective and unwantedcrosslinked molecules are obtained.

[0154] Preferred useful linking groups are derived from variousheterobifunctional cross-linking reagents such as those listed in thePierce Chemical Company Immunotechnology Catalog—Protein ModificationSection, (1995 and 1996). Useful non-limiting examples of such reagentsinclude:

[0155] Sulfo-SMCC Sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate.

[0156] Sulfo-SIAB Sulfosuccinimidyl (4-iodoacetyl)aminobenzoate.

[0157] Sulfo-SMPB Sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate.

[0158] 2-IT 2-Iminothiolane.

[0159] SATA N-Succinimidyl S-acetylthioacetate.

[0160] In addition to the foregoing description, the linking groups, inwhole or in part, can also be comprised of and derived fromcomplementary sequences of nucleotides and residues of nucleotides, bothnaturally occurring and modified, preferably non-self-associatingoligonucleotide sequences. Particularly useful, non-limiting reagentsfor incorporation of modified nucleotide moieties containing reactivefunctional groups, such as amine and sulfhydryl groups, into anoligonucleotide sequence are commercially available from, for example,Clontech Laboratories Inc. (Palo Alto Calif.) and include Uni-LinkAminoModifier (Catalog # 5190), Biotin-ON phosphoramidite (Catalog #5191), N-MNT-C6-AminoModifier (Catalog # 5202), AminoModifier II(Catalog # 5203), DMT-C6-3′Amine-ON (Catalog # 5222), C6-ThiolModifier(Catalog # 5211), and the like. In one aspect, linking groups of thisinvention are derived from the reaction of a reactive functional groupsuch as an amine or sulfhydryl group as are available in the aboveClontech reagents, one or more of which has been incorporated into anoligonucleotide sequence, with, for example, one or more of thepreviously described vector reactive groups such as a heterobifunctionalgroup on the vector.

[0161] By attaching two complementary oligonucleotide sequences one tothe vector and the other to the reporter the resulting double-strandedhybridized oligonucleotide then comprises the linking group between thevector and reporter.

[0162] Other polymer systems that serve as linkers include:—

[0163] Poly(L or D or DL-amino acids)=proteins and peptides; naturallyoccuring or synthetic

[0164] Pseudo Poly(amino acids)=(amino acids linked by non-amide bonds)

[0165] Poly (L or D or DL-lactide) and the co-polymers e.g Poly (L-lactide/DL -lactide)Poly (glycolide)

[0166] L-lactide/glycolide co-polymers

[0167] Poly-caprolactone and its co-polymers

[0168] Polyanhydrides

[0169] Poly (ortho esters)

[0170] Polyphosphazenes

[0171] Long-chain straight or branched lipids (& phospholipids)

[0172] Sugars and carbohydrates

[0173] Oligonucleotides (see above)

[0174] as well as mixtures of the above.

[0175] Linking agents used in accordance with the invention will ingeneral bring about linking of vector to reporter or reporter toreporter with some degree of specificity, and may also be used to attachone or more therapeutically active agents.

[0176] The present invention accordingly provides a tool for therapeuticdrug delivery in combination with vector-mediated direction of theproduct to the desired site. By “therapeutic” or “drug” is meant anagent having a beneficial effect on a specific disease in a living humanor non-human animal.

[0177] Therapeutic compounds used in accordance with the presentinvention may be encapsulated in the interior of a molecular aggregateor particulate linker or attached to or incorporated in theencapsulating walls of a vesicular linker. Thus, the therapeuticcompound may be linked to a part of the surface, for example throughcovalent or ionic bonds, or may be physically mixed into anencapsulating material, particularly if the drug has similar polarity orsolubility to the material, so as to prevent it from leaking out of theproduct before it is intended to act in the body. The release of thedrug may be initiated merely by wetting contact with blood followingadministration or as a consequence of other internal or externalinfluences, e.g. dissolution processes catalyzed by enzymes or the useof magnetic heating where the reporter is a magnetic particle.

[0178] The therapeutic substance may be covalently linked to theencapsulating membrane surface of a vascular linker using a suitablelinking acent, e.g. as described herein. Thus, for example, one mayinitially prepare a phospholipid derivative to which the drug is bondedthrough a biodegradable bond or linker, and then incorporate thisderivative into the material used to prepare the vesicle membrane, asdescribed above.

[0179] Alternatively, the agent may initially be prepared without thetherapeutic, which may then be coupled to or coated onto particulate(eg. vesicular) agents prior to use. Thus, for example, a therapeuticcould be added to a suspension of liposomes in aqueous media and shakenin order to attach or adhere the therapeutic to the liposomes.

[0180] The therapeutic may for example be a drug or prodrug known foruse in combatting angiogenesis or tumors.

[0181] By targeting an agent according to the invention containing aprodrug-activating enzyme to areas of pathology one may image targetingof the enzyme, making it possible to visualise when the agent istargeted properly and when the agent has disappeared from non-targetareas. In this way one can determine the optimal time for injection ofprodrug into individual patients.

[0182] Another alternative is to incorporate a prodrug, aprodrug-activating enzyme and a vector in the same particulate linkerreporter in such a way that the prodrug will only be activated aftersome external stimulus. Such a stimulus may, for example, be lightstimulation of a chromophoric reporter, or magnetic heating of asuperparamagnetic reporter after the desired targeting has beenachieved.

[0183] So-called prodrugs may also be used in agents according to theinvention. Thus drugs may be derivatised to alter their physicochemicalproperties and to adapt agent of the invention; such derivatised drugsmay be regarded as prodrugs and are usually inactive until cleavage ofthe derivatising group regenerates the active form of the drug.

[0184] Therapeutics may easily be delivered in accordance with theinvention to sites of angiogenesis.

[0185] By way of example, where the reporter is a chelated metal species(eg. a paramagnetic metal ion or a metal radionuclide), the linker maycomprise a chain attached to a metal chelating group, a polymeric chainwith a plurality of metal chelating groups pendant from the molecularbackbone or incorporated in the molecular backbone, a branched polymerwith metal chelating groups at branch termini (eg. a dendrimericpolychelant), etc. What is required of the linker is simply that it bindthe vector and reporter moieties together for an adequate period. Byadequate period is meant a period sufficient for the contrast agent toexert its desired effects, eg. to enhance contrast in vivo during adiagnostic imaging procedure.

[0186] Thus, in certain circumstances, it may be desirable that thelinker biodegrade after administration. By selecting an appropriatelybiodegradable linker it is possible to modify the biodistribution andbioelimination patterns for the vector and/or reporter. Where vectorand/or reporter are biologically active or are capable of exertingundesired effects if retained after the imaging procedure is over, itmay be desirable to design in linker biodegradability which ensuresappropriate bioelimination or metabolic breakdown of the vector and/orreporter moieties. Thus a linker may contain a biodegradable functionwhich on breakdown yields breakdown products with modifiedbiodistribution patterns which result from the release of the reporterfrom the vector or from fragmentation of a macromolecular structure. Byway of example for linkers which carry chelated metal ion reporters itis possible to have the linker incorporate a biodegradable functionwhich on breakdown releases an excretable chelate compound containingthe reporter.

[0187] Accordingly, biodegradable functions may if desired beincorporated within the linker structure, preferably at sites which are(a) branching sites, (b) at or near attachment sites for vectors orreporters, or (c) such that biodegradation yields physiologicallytolerable or rapidly excretable fragments.

[0188] Examples of suitable biodegradable functions include ester,amide, double ester, phosphoester, ether, thioether, guanidyl, acetaland ketal functions.

[0189] As discussed above, the linker group may if desired have builtinto its molecular backbone groups which affect the biodistribution ofthe contrast agent or which ensure appropriate spatial conformation forthe contrast agent, eg. to allow water access to chelated paramagneticmetal ion reporters. By way of example the linker backbone may consistin part or essentially totally of one or more polyalkylene oxide chains.

[0190] Thus the linker may be viewed as being a composite of optionallybiodegradable vector binding (V_(b)) and reporter binding (R_(b)) groupsjoined via linker backbone (L_(b)) groups, which linker backbone groupsmay carry linker side chain (L_(sc)) groups to modify biodistributionetc. and may themselves incorporate biodegradable functions. The R_(b)and V_(b) binding groups may be pendant from the linker backbone or maybe at linker backbone termini, for example with one R_(b) or V_(b) groupat one L_(b) terminus, with R_(b) or V_(b) groups linking together twoL_(b) termini or with one L_(b) terminus carrying two or more R_(b) orV_(b) groups. The L_(b) and L_(sc) groups will conveniently beoligomeric or polymeric structures (eg. polyesters, polyamides,polyethers, polyamines, oligopeptides, polypeptides, oligo andpolysaccharides, oligonucleotides, etc.), preferably structures havingat least in part a hydrophilic or lipophilic nature, eq. hydrophilic,amphiphilic or lipophilic structures.

[0191] The linker may be low, medium or high molecular weight, eq. up to2MD. Generally higher molecular weight linkers will be preferred if theyare to be loaded with a multiplicity of vectors or reporters or if it isnecessary to space vector and reporter apart, or if the linker is itselfto serve a role in the modification of biodistribution. In generalhowever linkers will be from 100 to 100 000 D, especially 120 D to 20 kDin molecular weight.

[0192] Conjugation of linker to vector and linker to reporter may be byany appropriate chemical conjugation technique, eq. covalent bonding(for example ester or amide formation), metal chelation or other metalcoordinative or ionic bonding, again as described above.

[0193] Examples of suitable linker systems include the magnifierpolychelant structures of U.S. Pat. No. 5,364,613 and PCT/EP90/00565,polyaminoacids (eg. polylysine), functionalised PEG, polysaccharides,glycosaminoglycans, dendritic polymers such as described in WO93/06868and by Tomalia et al. in Angew. Chem. Int. Ed. Engl. 29:138-175 (1990),PEG-chelant polymers such as described in W94/08629, WO94/09056 andWO96/26754, etc.

[0194] Where the reporter is a chelated metal ion, the linker group willgenerally incorporate the chelant moiety. Alternatively, the chelatedmetal may be carried on or in a particulate reporter. In either case,conventional metal chelating groups such as are well known in the fieldsof radiopharmaceuticals and MRI contrast media may be used, eg. linear,cyclic and branched polyamino-polycarboxylic acids and phosphorusoxyacid equivalents, and other sulphur and/or nitrogen ligands known inthe art, eg. DTPA, DTPA-BMA, EDTA, D03A, TMT (see for example U.S. Pat.No. 5,367,080), BAT and analogs (see for example Ohmono et al., J. Med.Chem. 35: 157-162 (1992) and Kung et al. J. Nucl. Med. 25: 326-332(1984)), the N₂S₂ chelant ECD of Neurolite, MAG (see Jurisson et al.Chem. Rev. 93: 1137-1156 (1993)), HIDA, DOXA(1-oxa-4,7,10-triazacyclododecanetriacetic acid), NOTA(1,4,7-triazacyclononanetriacetic acid), TETA(1,4,8,11-tetraazacyclotetradecanetetraacetic. acid), THT4′-(3-amino-4-methoxy-phenyl)-6,6″-bis(N′,N′-dicarboxymethyl-N-methylhydrazino)-2,2′:6′,2″-terpyridine),etc. In this regard, the reader is referred to the patent literature ofSterling Winthrop, Nycomed (including Nycomed Imaging and NycomedSalutar), Schering, Mallinckrodt, Bracco and Squibb relating tochelating agents for diagnostic metals, eg. in MR, X-ray andradiodiagnostic agents. See for example U.S. Pat. No. 4,647,447,EP-A-71564, U.S. Pat. No. 4,687,659, WO89/00557, U.S. Pat. No.4,885,363, and EP-A-232751.

[0195] Reporter

[0196] The reporter moieties in the contrast agents of the invention maybe any moiety capable of detection either directly or indirectly in anin vivo diagnostic imaging procedure, eg. moieties which emit or may becaused to emit detectable radiation (eg. by radioactive decay,fluorescence excitation, spin resonance excitation, etc.), moietieswhich affect local electromagnetic fields (eg. paramagnetic,superparamagnetic, ferrimagnetic or ferromagnetic species), moietieswhich absorb or scatter radiation energy (eg. chromophores andfluorophores), particles (including liquid containing vesicles), heavyelements and compounds thereof, and moieties which generate a detectablesubstance, etc.

[0197] A very wide range of materials detectable by diagnostic imagingmodalities is known from the art and the reporter will be selectedaccording to the imaging modality to be used. Thus for example forultrasound imaging an echogenic material, or a material capable ofgenerating an echogenic material will normally be selected, for X-rayimaging the reporter will generally be or contain a heavy atom (eq. ofatomic weight 38 or above), for MR imaging the reporter will either be anon zero nuclear spin isotope (such as ¹⁹F) or a material havingunpaired electron spins and hence paramagnetic, superparamagnetic,ferrimagnetic or ferromagnetic properties, for light imaging thereporter will be a light scatterer (eq. a coloured or uncolouredparticle), a light absorber or a light emitter, for magnetometricimaging the reporter will have detectable magnetic properties, forelectrical impedance imaging the reporter will affect electricalimpedance and for scintigraphy, SPECT, PET etc. the reporter will be aradionuclide.

[0198] Examples of suitable reporters are widely known from thediagnostic imaging literature, eg. magnetic iron oxide particles, X-raycontrast agent containing vesicles, chelated paramagnetic metals (suchas Gd, Dy, Mn, Fe etc.). See for example U.S. Pat. No. 4,647,447,PCT/GB97/00067, U.S. Pat. No. 4,863,715, U.S. Pat. No. 4,770,183,WO96/09840, WO85/02772, WO92/17212, PCT/GB97/00459, EP-A-554213, U.S.Pat. No. 5,228,446, WO91/15243, WO93/05818, WO96/23524, WO96/17628, U.S.Pat. No. 5,387,080, WO95/26205, GB9624918.0, etc.

[0199] Particularly preferred as reporters are: chelated paramagneticmetal ions such as Gd, Dy, Fe, and Mn, especially when chelated bymacrocyclic chelant groups (eg. tetraazacyclododecane chelants such asDOTA, DO3A, HP-DO3A and analogues thereof) or by linker chelant groupssuch as DTPA, DTPA-BMA, EDTA, DPDP, etc; metal radionuclide such as ⁹⁰Y,^(99m)Tc, ¹¹¹In, ⁴⁷Sc, ⁶⁷/Ga, ⁵¹Cr, ^(177m)Sn, ⁶⁷Cu, ¹⁶⁷Tm, ⁹⁷Ru, 188Re,¹⁷⁷Lu, ¹⁹⁹Au, ²⁰³Pb and ¹⁴¹Ce; superparamagnetic iron oxide crystals;chromophores and fluorophores having absorption and/or emission maximain the range 300-1400 nm, especially 600 nm to 1200 nm, in particular650 to 1000 nm; chelated heavy metal cluster ions (eg. W or Mopolyoxoanions or the sulphur or mixed oxygen/sulphur analogs);covalently bonded non-metal atoms which are either high atomic number(eg. iodine) or are radioactive, eg ¹²³I, ¹³¹I, etc. atoms; iodinatedcompound containing vesicles; etc.

[0200] Stated generally, the reporter may be (1) a chelatable metal orpolyatomic metal-containing ion (ie. TcO, etc), where the metal is ahigh atomic number metal (eg. atomic number greater than 37), aparamagentic species (eg. a transition metal or lanthanide), or aradioactive isotope, (2) a covalently bound non-metal species which isan unpaired electron site (eg. an oxygen or carbon in a persistant freeradical), a high atomic number non-metal, or a radioisotope, (3) apolyatomic cluster or crystal containing high atomic number atoms,displaying cooperative magnetic behaviour (eg. superparamagnetism,ferrimagnetism or ferromagnetism) or containing radionuclides, (4) achromophore (by which term species which are fluorescent orphosphorescent are included), eg. an inorganic or organic structure,particularly a complexed metal ion or an organic group having anextensive delocalized electron system, or (5) a structure or grouphaving electrical impedance varying characteristics, eg. by virtue of anextensive delocalized electron system.

[0201] Examples of particular preferred reporter groups are described inmore detail below.

[0202] Chelated metal reporters: metal radionuclides, paramagnetic metalions, fluorescent metal ions, heavy metal ions and cluster ions

[0203] Preferred metal radionuclides include 90Y, ^(99m)Tc, ¹¹¹In, ⁴⁷SC,⁶⁷Ga, ⁵¹Cr, ^(177m)Sn, ⁶⁷Cu, ¹⁶⁷Tm, ⁹⁷Ru, ¹⁸⁸ Re, ¹⁷⁷Lu, ¹⁹⁹Au, ²⁰³Pband ¹⁴¹Ce.

[0204] Preferred paramagnetic metal ions include ions of transition andlanthanide metals (eg. metals having atomic numbers of 6 to 9, 21-29,42, 43, 44, or 57-71), in particular ions of Cr, V, Mn, Fe, Co, Ni, Cu,La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu,especially of Mn, Cr, Fe, Gd and Dy, more especially Gd.

[0205] Preferred fluorescent metal ions include lanthanides, inparticular La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, andLu. Eu is especially preferred.

[0206] Preferred heavy metal-containing reporters may include atoms ofMo, Bi, Si, and W, and in particular may be polyatomic cluster ions (eg.Bi compounds and W and Mo oxides) as described in WO91/14460,WO92/17215, WO96/40287, and WO96/22914.

[0207] The metal ions are desirably chelated by chelant groups on thelinker moiety or in or on a particle, (eg. a vesicle or a porous ornon-porous inorganic or organic solid), in particular linear,macrocyclic, terpyridine and N₂S₂ chelants, such as for example DTPA,DTPA-BMA, EDTA, D03A, TMT. Further examples of suitable chelant groupsare disclosed in U.S. Pat. No. 4,647,447, WO89/00557, US-A-5367080, U.S.Pat. No. 5,364,613, etc.

[0208] The linker moiety or the particle may contain one or more suchchelant groups, if desired metallated by more than one metal species(eg. so as to provide reporters detectable in different imagingmodalities).

[0209] Particularly where the metal is non-radioactive, it is preferredthat a polychelant linker or particulate reporter be used.

[0210] A chelant or chelating group as referred to herein may comprisethe residue of one or more of a wide variety of chelating agents thatcan complex a metal ion or a polyatomic ion (eg. TcO).

[0211] As is well known, a chelating agent is a compound containingdonor atoms that can combine by coordinate bonding with a metal atom toform a cyclic structure called a chelation complex or chelate. Thisclass of compounds is described in the Kirk-Othmer Encyclopedia ofChemical Technology, Vol. 5, 339-368.

[0212] The residue of a suitable chelating agent can be selected frompolyphosphates, such as sodium tripolyphosphate and hexametaphosphoricacid; aminocarboxylic acids, such as ethylenediaminetetraacetic acid,N-(2-hydroxy)ethylene-diaminetriacetic acid, nitrilotriacetic acid,N,N-di(2-hydroxyethyl)glycine, ethylenebis(hydroxyphenylglycine) anddiethylenetriamine pentacetic acid; 1,3-diketones, such asacetylacetone, trifluoroacetylacetone, and thenoyltrifluoroacetone;hydroxycarboxylic acids, such as tartaric acid, citric acid, gluconicacid, and 5-sulfosalicyclic acid; polyamines, such as ethylenediamine,diethylenetriamine, triethylenetetraamine, and triaminotriethylamine;aminoalcohols, such as triethanolamine andN-(2-hydroxyethyl)ethylenediamine; aromatic heterocyclic bases, such as2,2′-diimidazole, picoline amine, dipicoline amine and1,10-phenanthroline; phenols, such as salicylaldehyde,disulfopyrocatechol, and chromotropic acid; aminophenols, such as8-hydroxyquinoline and oximesulfonic acid; oximes, such asdimethylglyoxime and salicylaldoxime; peptides containing proximalchelating functionality such as polycysteine, polyhistidine,polyaspartic acid, polyglutamic acid, or combinations of such aminoacids; Schiff bases, such as disalicylaldehyde 1,2-propylenediimine;tetrapyrroles, such as tetraphenylporphin and phthalocyanine; sulfurcompounds, such as toluenedithiol, meso-2,3-dimercaptosuccinic acid,dimercaptopropanol, thioglycolic acid, potassium ethyl xanthate, sodiumdiethyldithiocarbamate, dithizone, diethyl dithiophosphoric acid, andthiourea; synthetic macrocyclic compounds, such as dibenzo[18]crown-6,(CH₃)₆-[14]-4,11]-diene-N₄, and (2.2.2-cryptate); phosphonic acids, suchas nitrilotrimethylene-phosphonic acid,ethylenediaminetetra(methylenephosphonic acid), andhydroxyethylidenediphosphonic acid, or combinations of two or more ofthe above agents. The residue of a suitable chelating agent preferablycomprises a polycarboxylic acid group and preferred examples include:ethylenediamine-N,N,N′,N′-tetraacetic acid (EDTA);N,N,N′,N″,N″-diethylene-triaminepentaacetic acid (DTPA);1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA);1,4,7,10-tetraazacyclododecane-N,N′,N″-triacetic acid (DOTA);1-oxa-4,7,10-triazacyclododecane-N,N′,N′″-triacetic acid (OTTA);trans(1,2)-cyclohexanodiethylene-triamine-pentaacetic acid (CDTPA).

[0213] Other suitable residues of chelating agents comprise proteinsmodified for the chelation of metals such as technetium and rhenium asdescribed in U.S. Pat. No. 5,078,985, the disclosure of which is herebyincorporated by reference.

[0214] Suitable residues of chelating agents may also derive from N3Sand N2S2 containing compounds, as for example, those disclosed in U.S.Pat. Nos. 4,444,690; 4,670,545; 4673562; 4897255; 4965392; 4980147;4988496; 5021556 and 5075099.

[0215] Other suitable residues of chelating are described inPCT/US91/08253, the disclosure of which is hereby. incorporated byreference.

[0216] Preferred chelating groups are selected from the group consistingof 2-amiomethylpyridine, iminoacetic acid, iminodiacetic acid,ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid(DOTA), carbonyliminodiacetic acid, methyleneiminoacetic acid,methyleneiminodiacetic acid, ethylenethioethylene-iminoacetic acid,ethylenethioethyleneiminodiacetic acid, TMT, a terpyridinyl group, achelating agent comprising a terpyridyl group and a carboxymethylaminogroup, or a salt of any of the foregoing acids. Especially preferredchelating groups are DTPA, DTPA-BMA, DPDP, TMT, DOTA and HPDO3A.

[0217] Representative chelating groups are also described in U.S. Pat.No. 5,559,214 A, WO 9526754, WO 9408624, WO 9409056, WO 9429333, WO9408624, WO 9408629 A1, WO 9413327 A1 and WO 9412216 A1.

[0218] Methods for metallating any chelating agents present are withinthe level of skill in the art. Metals can be incorporated into a chelantmoiety by any one of three general methods: direct incorporation,template synthesis and/or transmetallation. Direct incorporation ispreferred.

[0219] Thus it is desirable that the metal ion be easily complexed tothe chelating agent, for example, by merely exposing or mixing anaqueous solution of the chelating agent-containing moiety with a metalsalt in an aqueous solution preferably having a pH in the range of about4 to about 11. The salt can be any salt, but preferably the salt is awater soluble salt of the metal such as a halogen salt, and morepreferably such salts are selected so as not to interfere with thebinding of the metal ion with the chelating agent. The chelatingagent-containing moiety is preferrably in aqueous solution at a pH ofbetween about 5 and about 9, more preferably between pH about 6 to about8. The chelating agent-containing moiety can be mixed with buffer saltssuch as citrate, acetate, phosphate and borate to produce the optimumpH. Preferably, the buffer salts are selected so as not to interferewith the subsequent binding of the metal ion to the chelating agent.

[0220] In diagnostic imaging, the vector-linker-reporter (VLR) constructpreferably contains a ratio of metal radionuclide ion to chelating agentthat is effective in such diagnostic imaging applications. In preferredembodiments, the mole ratio of metal ion per chelating agent is fromabout 1:1,000 to about 1:1.

[0221] In radiotherapeutic applications, the VLR preferably contains aratio of metal radionuclide ion to chelating agent that is effective insuch therapeutic applications. In preferred embodiments, the mole ratioof metal ion per chelating agent is from about 1:100 to about 1:1. Theradionuclide can be selected, for example, from radioisotopes of Sc, Fe,Pb, Ga, Y, Bi, Mn, Cu, Cr, Zn, Ge, Mo, Ru, Sn, Sr, Sm, Lu, Sb, W, Re,Po, Ta and Tl. Preferred radionuclides include ⁴⁴Sc, ⁶⁴Cu, ⁶⁷Cu, 212Pb,⁶⁸Ga, ⁹⁰Y, ¹⁵³Sm, ²¹²Bi, ¹⁸⁶Re and ¹⁸⁸Re. Of these, especially preferredis ⁹⁰Y. These radioisotopes can be atomic or preferably ionic.

[0222] The following isotopes or isotope pairs can be used for bothimaging and therapy without having to change the radiolabelingmethodology or chelator: ⁴⁷Sc₂₁; ¹⁴¹Ce₅₈; ¹⁸⁸Re₇₅; ¹⁷⁷Lu₇₁; ¹⁹⁹Au₇₉;⁴⁷Sc₂₁; ¹³¹I₅₃; ⁶⁷Cu₂₉; ¹³¹I₅₃ and ¹²³I₅₃; ¹⁸⁸Re₇₅ and ^(99m)Tc₄₃; ⁹⁰Y₃₉and ⁸⁷Y₃₉; ⁴⁷Sc₂₁ and ⁴⁴Sc₂₁; ⁹⁰Y₃₉ and ¹²³I_(53;) ¹⁴⁶Sm₆₂ and ¹⁵³Sm₆₂;and ⁹⁰Y₃₉ and ¹¹¹In₄₉.

[0223] Where the linker moiety contains a single chelant, that chelantmay be attached directly to the vector moiety, eg. via one of the metalcoordinating groups of the chelant which may form an ester, amide,thioester or thioamide bond with an amine, thiol or hydroxyl group onthe vector. Alternatively the vector and chelant may be directly linkedvia a functionality attached to the chelant backbone, eg. aCH₂-phenyl-NCS group attached to a ring carbon of DOTA as proposed byMeares et al. in JACS 110:6266-6267(1988), or indirectly via a homo orhetero-bifunctional linker, eg. a bis amine, bis epoxide, diol, diacid,difunctionalised PEG, etc. In that event, the bifunctional linker willconveniently provide a chain of 1 to 200, preferably 3 to 30 atomsbetween vector and chelant residue.

[0224] Where the linker moiety contains a plurality of chelant groups,the linker preferably is or contains portions of formula

[0225] where Ch is a chelant moiety and Li is a linker backbonecomponent, ie. the linker preferably has pendant chelants, in-backbonechelants or terminal chelants or a combination thereof. The pendant andin-backbone polymeric structures may be branched but more preferably arelinear and the repeat units (LiCh) or other repeat units in the polymermay have in-backbone or pendant biodistribution modifying groups, eg.polyalkylene groups as in WO94/08629, WO94/09056, and WO96/20754. Theterminal chelant structures Li(Ch), which may be dendritic polymers asin WO93/06868, may have biodistribution modifying groups attached totermini not occupied by chelants and may have biodegradation enhancingsites within the linker structure as in WO95/28966.

[0226] The chelant moieties within the polychelant linker may beattached via backbone functionalization of the chelant or by utilizationof one or more of the metal co-ordinating groups of the chelant or byamide or ether bond formation between acid chelant and an amine orhydroxyl carrying linker backbone, eg. as in polylysine-polyDTPA,polylysine-polyDOTA and in the so-called magnifier polychelants, ofPCT/EP96/00565. Such polychelant linkers may be conjugated to one ormore vector groups either directly (eg. utilizing amine, acid orhydroxyl groups in the polychelant linker) or via a bifunctional linkercompound as discussed above for monochelant linkers.

[0227] Where the chelated species is carried by a particulate (ormolecular aggregate, eg. vesicular) linker, the chelate may for examplebe an unattached mono or polychelate (such as Gd DTPA-BMA or Gd HP-DO3A)enclosed within the particle or it may be a mono or polychelateconjugated to the particle either by covalent bonding or by interactionof an anchor group (eg. a lipophilic group) on the mono/polychelate withthe membrane of a vesicle (see for example PCT/GB95/02378).

[0228] Non-Metal Atomic Reporters

[0229] Preferred non-metal atomic reporters include radioisotopes suchas ¹²³I and ¹³¹I as well as non zero nuclear spin atoms such as ¹⁹F, andheavy atoms such as I.

[0230] Such reporters, preferably a plurality thereof, eg. 2 to 200, maybe covalently bonded to a linker backbone, either directly usingconventional chemical synthesis techniques or via a supporting group,eg. a triiodophenyl group.

[0231] In an embodiment of this invention, the use of radioisotopes ofiodine is specifically contemplated. For example, if the vector orlinker is comprised of substituents that can be chemically substitutedby iodine in a covalent bond forming reaction, such as, for example,substituents containing hydroxyphenyl functionality, such substituentscan be labeled by methods well known in the art with a radioisotope ofiodine. The iodine species can be used in therapeutic and diagnosticimaging applications. While, at the same time, a metal in a chelatingagent on the same vector-linker can also be used in either therapeuticor diagnostic imaging applications.

[0232] As with the metal chelants discussed above, such metal atomicreporters may be linked to the linker or carried in or on aparticulate-linker, eg. in a vesicle (see WO95/26205 and GB9624918.0).

[0233] Linkers of the type described above in connection with the metalreporters may be used for non-metal atomic reporters with the non-metalatomic reporter or groups carrying such reporters taking the place ofsome or all of the chelant groups.

[0234] Organic Chromophoric or Fluorophoric Reporters

[0235] Preferred organic chromophoric and fluorophoric reporters includegroups having an extensive delocalized electron system, eg. cyanines,merocyanines, phthalocyanines, naphthalocyanines, triphenylmethines,porphyrins, pyrilium dyes, thiapyrilium dyes, squarylium dyes, croconiumdyes, azulenium dyes, indoanilines, benzophenoxazinium dyes,benzothiaphenothiazinium dyes, anthraquinones, napthoquinones,indathrenes, phthaloylacridones, trisphenoquinones, azo dyes,intramolecular and intermolecular charge-transfer dyes and dyecomplexes, tropones, tetrazines, bis(dithiolene) complexes,bis(benzene-dithiolate) complexes, iodoaniline dyes, bis(S,O-dithiolene)complexes, etc. Examples of suitable organic or metallated organicchromophores may be found in “Topics in Applied Chemistry: Infraredabsorbing dyes” Ed. M. Matsuoka, Plenum, N.Y. 1990, “Topics in AppliedChemistry: The Chemistry and Application of Dyes”, Waring et al.,Plenum, N.Y., 1990, “Handbook of Fluorescent Probes and ResearchChemicals” Haugland, Molecular Probes Inc, 1996, DE-A-4445065,DE-A-4326466, JP-A-3/228046, Narayanan et al. J. Org. Chem. 60:2391-2395 (1995), Lipowska et al. Heterocyclic Comm. 1: 427-430 (1995),Fabian et al. Chem. Rev. 92: 1197 (1992), WO96/23525, Strekowska et al.J. Org. Chem. 57: 4578-4580 (1992), WO (Axis) and WO96/17628. Particularexamples of chromophores which may be used include xylene cyanole,fluorescein, dansyl, NBD, indocyanine green, DODCI, DTDCI, DOTCI andDDTCI.

[0236] Particularly preferred are groups which have absorption maximabetween 600 and 1000 nm to avoid interference with haemoglobinabsorption (eg. xylene cyanole).

[0237] Further such examples include:

[0238] cyanine dyes: such as heptamethinecyanine dyes, e.g. compounds 4ato 4 g Table II on page 26 of Matsuoka (supra)

4a: where Y = S, X = I, R = Et 4b: where Y = S, X = ClO₄, R = Et 4c:where Y = Cme₂, X = I, R = Me 4d: where Y = CMe₂, X = ClO₄, R = Me 4e:where Y = CH═CH, X = I, R = Et 4f: where Y = CH═CH, X = Br, R = Et 4g:where Y = CH═CH, X = ClO₄, R = Et

[0239] and in Table III on page 28 of Matsuoka (supra), i.e.

where Y = O, X = I, R = Me where Y = CMe₂, X = I, R = Me where Y = S, X= Br R = Et;

[0240] chalcogenopyrylomethine dyes, e.g., compounds 12 on page 31 ofMatsuoka (supra), i.e.

[0241] where Y═Te, Se, O or NR;

[0242] monochalcogenopyrylomethine dyes, e.g. compounds 13 on page 31,of Matsuoka (supra) i.e.

[0243] where n=1 or 2;

[0244] pyrilium dyes, e.g., compounds 14 (X═O) on page 32 of Matsuoka(supra), i.e.

[0245] where X O, S, or Se;

[0246] thiapyrilium dyes, e.g. compounds 15 on page 32, and compound Ion page 167 of Matsuoka (supra), i.e.

[0247] where n 1 or 2;

[0248] squarylium dyes, e.g. compound 10 and Table IV on page 30 ofMatsuoka (supra), i.e.

[0249] where X═CH═CH, Y═H, and R═Et,

[0250] X═S, Y═H, and R═Et, and

[0251] X═CMe₂, Y═H, and R═Me,

[0252] and compound 6, page 26, of Matsuoka (supra), i.e.

[0253] where X═CH═CH, Y H, and R═Et;

[0254] croconium dyes, e.g. compound 9 and Table IV on page 30 ofMatusoka (supra), i.e.

[0255] where X═CH═CH, Y═H, and R═Et,

[0256] X═S, Y═H, and R═Et,

[0257] X═CMe₂, Y═H, and R═Me,

[0258] and compound 7, page 26, of Matsuoka (supra), i.e.

[0259] where X═CH═CH, Y═H, and R═Et;

[0260] azulenium dyes, e.g. compound 8 on page 27 of Matsuoka (supra),i.e.

[0261] merocyanine dyes, e.g. compound 16, R═Me, on page 32 of Matsuoka(supra), i.e.

[0262] indoaniline dyes such as copper and nickel complexes ofindoaniline dyes, e.g. compound 6 on page 63 of Matsuoka (supra), i.e.

[0263] where R═Et, R′═Me, M═Cu,

[0264] R═Et, R′═Me, M═Ni,

[0265] R═Me, R′═H, M═Cu, or

[0266] R═Me, R¹═H, M═Ni,

[0267] benzo[a]phenoxazinium dyes and benzo[a]phenothiazinium dyes, e.g.as shown on page 201 of Matusoka (supra), i.e.

[0268] where X═O or S;

[0269] 1,4-diaminoanthraquinone(N-alkyl)-3′-thioxo-2,3-dicarboximides,e.g. compound 20, on page 41 of Matusoka (supra)

[0270] indanthrene pigments, e.g.

[0271] see compound 21 on page 41 of Matsuoka (supra);2-arylamino-3,4-phthaloylacridone dyes, e.g. compound 22 on page 41 ofMatsuoka (supra)

[0272] trisphenoquinone dyes, e.g. compound 23 on page 41 of Matsuoka(supra)

[0273] azo dyes, e.g. the monoazo dye, compound 2 on page 90 of Matsuoka(supra), i.e.

[0274] where X═CH═C(CN)₂, R₁═R₂═Et, R₃, R₄═H,

[0275] X═C(CN)⊚C(CN)₂, R₁═R₂═Et, R₃═R₄═H, or

[0276] X═

[0277] and Y═C═O, R₁═R₂═Et, R₃═R₄═H, or Y═SO₂, R₁═H, R₂═CH(Me)nBu,R₃═OMe, and R₄═NHAC;

[0278] azo dyes, e.g. the polyazo dye, compound 5 on page 91 of Matsuoka(supra), i.e.

[0279] intramolecular charge transfer donor-acceptor infrared dyes, e.g.compounds 6 and 7 on page 91 of Matsuoka (supra), i.e.

[0280] and

[0281] nonbenzenoid aromatic dyes, e.g. compound 8, a tropone, on page92, of Matsuoka (supra), i.e.

[0282] tetrazine radical dyes, e.g. compound 9 on page 92 of Matsuoka(supra), i.e.

[0283] in which, X=p-phenylene or

[0284] X=p-terphenylene as well as compound 10 on page 92 of Matsuoka(supra), i.e.

[0285] in which X=p-biphenyl;

[0286] cationic salts of tetrazine radical dyes, e.g. compound 11 onpage 92 of Matsuoka (supra)

[0287] in which X=p-phenylene;

[0288] donor-acceptor intermolecular charge transfer dyes, e.g. CTcomplexes of compounds 13b and 14a to 14c on page 93 of Matsuoka(supra), i.e.

[0289] where X CH═N—N(Ph)₂ in the donor and

[0290] a) Y═CN, Z═NO₂

[0291] b) Y═CN, Z═H or

[0292] a) Y═Cl, Z═NO₂ in the acceptor;

[0293] anthraquinone dyes, e.g. compounds 12 (X═S or Se) on page 38 ofMatsuoka (supra), i.e.

[0294] wherein X═S or Se and Y=tetrachloro, tetrabromo, 2,3-dicarboxylicacid, 2,3-dicarboxylic anhydride, or 2,3-dicarboxylic acid N-phenylimide; naphthoquinone dyes, e.g. compounds 2, 3, and 4 on page 37, ofMatsuoka (supra), i.e.

[0295] metallated azo dyes such as azo dyes containing nickel, cobalt,copper, iron, and manganese;

[0296] phthalocyanine dyes, e.g. compound 1 in Table II on page 51 ofMatsuoka (supra), e.g.

[0297] naphthalocyanine dyes, e.g. compound 3 in Table II on page 51 ofMatsuoka (supra), e.g.

[0298] metal phthalocyanines such as phthalocyanines containingaluminum, silicon, nickel, zinc, lead, cadmium, magnesium, vanadium,cobalt, copper, and iron, e.g. compound 1 in Table III on page 52 ofMatsuoka (supra), e.g.

[0299] in which, for example, M═Mg;

[0300] metal naphthalocyanines such as naphthalocyanines containingaluminum, zinc, cobalt, magnesium, cadmium, silicon, nickel, vanadium,lead, copper, and iron, see compound 3 in Table III on page 52 ofMatsuoka (supra), e.g.

[0301] in which, for example, M═Mg;

[0302] bis(dithiolene) metal complexes comprising a metal ion such asnickel, cobalt, copper, and iron coordinated to four sulfur atoms in abis(S,S′-bidentate) ligand complex, e.g. see Table I on page 59 ofMatsuoka (supra)

[0303] where

[0304] R₁═R₂═CF₃, M═Ni,

[0305] R₁═R₂═phenyl, M═Pd,

[0306] R₁═R₂═phenyl, M═Pt,

[0307] R₁═C4 to C10 alkyl, R₂═H, M═Ni,

[0308] R₁═C4 to C10 alkyl, R₂═H, M═Pd,

[0309] R₁═C4 to C10 alkyl, R₂═H, M═Pt,

[0310] R₁═R₂=phenyl, M═Ni,

[0311] R₁═R₂=p-CH₃-phenyl, M═Ni,

[0312] R₁═R₂=p-CH₃O-phenyl, M═Ni,

[0313] R₁═R₂=p-Cl-phenyl, M═Ni,

[0314] R₁═R₂=p-CF₃-phenyl, M═Ni,

[0315] R₁═R₂=3,4,-diCl-phenyl, M═Ni,

[0316] R₁═R₂═o-Cl-phenyl, M═Ni,

[0317] R₁═R₂=o-Br-phenyl, M═Ni,

[0318] R₁═R₂=3,4,-diCl-phenyl, M═Ni,

[0319] R₁═R₂=p-CH₃, M═Ni,

[0320] R₁═R₂=2-thienyl, M═Ni,

[0321] R₁=p-(CH₃)₂ N-phenyl, R₂=phenyl, M═Ni, and

[0322] R₁=p-(CH₃)₂ N-phenyl, R₂=p-H₂N-phenyl, M═Ni;

[0323] bis(benzenedithiolate) metal complexes comprising a metal ionsuch as nickel, cobalt, copper, and iron coordinated to four sulfuratoms in a ligand complex, e.g. see Table III on page 62 of Matsuoka(supra), i.e.

[0324] where

[0325] X=tetramethyl, M═Ni,

[0326] X=4,5-dimethyl, M═Ni,

[0327] X=4-methyl, M═Ni,

[0328] X=tetrachloro, M═Ni,

[0329] X═H, M═Ni,

[0330] X=4-methyl, M═Co,

[0331] X=4-methyl, M═Cu, and

[0332] X=4-methyl, M═Fe;

[0333] N,O-bidentate indoaniline dyes comprising a metal ion such asnickel, cobalt, copper, and iron coordinated to two nitrogen and twooxygen atoms of two N,O-bidentate indoaniline ligands, e.g. compound 6in Table IV on page 63 of Matsuoka (supra), e.g.

[0334] where R═Et, R′═Me, M═Cu,

[0335] R═Et, R′═Me, M═Ni,

[0336] R═Me, R′═H, M═Cu, and

[0337] R═Me, R′═H, M═Ni,

[0338] bis(S,O-dithiolene) metal complexes comprising a metal ion suchas nickel, cobalt, copper, and iron coordinated to two sulfur atoms andtwo oxygen atoms in a bis(S,O-bidentate) ligand complex, e.g. see U.S.Pat. No. 3,806,462, e.g.

[0339] a-diimine-dithiolene complexes comprising a metal ion such asnickel, cobalt, copper, and iron coordinated to two sulfur atoms and twoimino-nitrogen atoms in a mixed S,S- and N,N-bidentate diligand complex,e.g. see Table II on page 180, second from bottom, of Matsuoka (supra)(also see Japanese patents: 62/39,682, 63/126,889 and 63/139,303), e.g.

[0340] and

[0341] tris(a-diimine) complexes comprising a metal ion coordinated tosix nitrogen atoms in a triligand complex, e.g. see Table II on page 180of Matsuoka (supra), last compound, (also see Japanese Patents 61/20,002and 61/73,902), e.g.

[0342] Representative examples of visible dyes include fluoresceinderivatives, rhodamine derivatives, coumarins, azo dyes, metalizabledyes, anthraquinone dyes, benzodifuranone dyes, polycyclic aromaticcarbonyl dyes, indigoid dyes, polymethine dyes, azacarbocyanine dyes,hemicyanine dyes, barbituates, diazahemicyanine dyes, stryrl dyes,diaryl carbonium dyes, triaryl carbonium dyes, phthalocyanine dyes,quinophthalone dyes, triphenodioxazine dyes, formazan dyes,phenothiazine dyes such as methylene blue, azure A, azure B, and azureC, oxazine dyes, thiazine dyes, naphtholactam dyes, diazahemicyaninedyes, azopyridone dyes, azobenzene dyes, mordant dyes, acid dyes, basicdyes, metallized and premetallized dyes, xanthene dyes, direct dyes,leuco dyes which can be oxidized to produce dyes with huesbathochromically shifted from those of the precursor leuco dyes, andother dyes such as those listed by Waring, D. R. and Hallas, G., in “TheChemistry and Application of Dyes”, Topics in Applied Chemistry, PlenumPress, New York, N.Y., 1990. Additonal dyes can be found listed inHaugland, R. P., “Handbook of Fluorescent Probes and ResearchChemicals”, Sixth Edition, Molecular Probes, Inc., Eugene Oreg., 1996.

[0343] Such chormophores and fluorophores, may be covalently linkedeither directly to the vector or to or within a linker structure. Onceagain linkers of the type described above in connection with the metalreporters may be used for organic chromophores or fluorophores with thechromophores/fluorophores taking the place of some or all of the chelantgroups.

[0344] As with the metal chelants discussed abovechromophores/fluorophores may be carried in or on a particulatelinker-moieties, eg. in or on a vesicle or covalently bonded to inertmatrix particles that can also function as a light scattering reporter.

[0345] Particulate Reporters or Linker-Reporters

[0346] The particulate reporters and linker-reporters generally fallinto two categories—those where the particle comprises a matrix or shellwhich carries or contains the reporter and those where the particlematrix is itself the reporter. Examples of the first category are:vesicles (eg. micelles and liposomes) containing a liquid or solid phasewhich contains the contrast effective reporter, eg. a chelatedparamagnetic metal or radionuclide, or a water-soluble iodinated X-raycontrast agent; porous particles loaded with the reporter, eg.paramagnetic metal loaded molecular sieve particles; and solidparticles, eg. of an inert biotolerable polymer, onto which the reporteris bound or coated, eg. dye-loaded polymer particles.

[0347] Examples of the second category are: light scattering organic orinorganic particles; magnetic particles (ie. superparamagnetic,ferromagnetic or ferrimagnetic particles); and dye particles.

[0348] Preferred particulate reporters or reporter-linkers includesuperparamagnetic particles (see U.S. Pat. No. 4,770,183,PCT/GB97/00067, WO96/09840, etc.), echogenic vesicles (see WO92/17212,PCT/GB97/00459, etc.), iodine-containing vesicles (see WO95/26205 andGB9624918.0), and dye-loaded polymer particles (see WO96/23524). Theparticulate reporters may have one or more vectors attached directly orindirectly to their surfaces. Generally it will be preferred to attach aplurality (eg. 2 to 50) of vector moieties per particle. Particularlyconveniently, besides the desired targeting vector, one will alsoattached flow decelerating vectors to the particles, ie. vectors whichhave an affinity for the capillary lumen or other organ surfaces whichis sufficient to slow the passage of the contrast agent through thecapillaries or the target organ but not sufficient on its own toimmobilise the contrast agent. Such flow decelerating vectors (describedfor example in GB9700699.3) may moreover serve to anchor the contrastagent once it has bound to its target site.

[0349] The means by which vector to particle attachment is achieved willdepend on the nature of the particle surface. For inorganic particles,the linkage to the particle may be for example by way of interactionbetween a metal binding group (eg. a phosphate, phosphonate or oligo orpolyphosphate group) on the vector or on a linker attached to thevector. For organic (eg. polymeric) particles, vector attachment may beby way of direct covalent bonding between groups on the particle surfaceand reactive groups in the vector, eg. amide or ester bonding, or bycovalent attachment of vector and particle to a linker. Linkers of thetype discussed above in connection with chelated metal reporters may beused although in general the linkers will not be used to coupleparticles together. For non-solid particles, eg. droplets (for exampleof water insoluble iodinated liquids as described in U.S. Pat. No.5,318,767, U.S. Pat. No. 5,451,393, U.S. Pat. No. 5,352,459 and U.S.Pat. No. 5,569,448) and vesicles, the linker may conveniently containhydrophobic “anchor” groups, for example saturated or unsaturated C₁₂₋₃₀chains, which will penetrate the particle surface and bind vector toparticle. Thus for phospholipid vesicles, the linker may serve to bindthe vector covalently to a phospholipid compatible with the vesiclemembrane. Examples of linker binding to vesicles and inorganic particlesare described in GB9622368.0 and PCT/GB97/00067.

[0350] Besides the vectors, other groups may be bound to the particlesurface, eg. stabilisers (to prevent aggregation) and biodistributionmodifiers such as PEG. Such groups are discussed for example inPCT/GB97/00067, WO96/09840, EP-A-284549 and US-A-4904479.

[0351] Preferably the V-L-R agents of the invention will have thereceptor targetting vectors coupled directly or indirectly to areporter, eg. with covalently bound iodine radioisotopes, with metalchelates attached directly or via an organic linker group or coupled toa particulate reporter or linker-reporter, eg. a superparamagneticcrystals (optionally coated, eg. as in PCT/GB97/00067), or a vesicle,e.g. an iodinated contrast agent containing micelle or liposome.

[0352] Put briefly, for the imaging modalities of MRI, X-ray, lightimaging, nuclear imaging, magnetotomography and electrical impedancetomography, the favoured reporters may be as follows: MRISuperparamgnetic iron oxide particles, in general having a particle sizesmaller than about 80 nm. In particular iron oxides coated with variouscoating materials such as polyelectrolytes, PEG, starch and hyrolyzedstarch are preferred. Paramagnetic metal substances including bothchelates and particulate materials are also useful. Light imaging Anylight imaging reporter group. The focus should be on substancesabsorbing in the near infrared range. Nuclear medicine Radioactivechelates comprising ⁹⁹Tc or ¹¹¹In as well as direct radiolabelledvectors having radiolabelled halogens substituents such as ¹²³I, ¹²⁵I,¹³¹I, ⁷⁵Br or ⁷⁷Br. Magnetotomography Superparmagnetic iron oxideparticles as described above. Electrical impedance Polyionic species,e.g. polymers tomography with ionic groups in the repeat units.

[0353] The agents of the invention may be administered to patients forimaging in amounts sufficient to yield the desired contrast with theparticular imaging technique. Where the reporter is a metal, generallydosages of from 0.001 to 5.0 mmoles of chelated imaging metal ion perkilogram of patient bodyweight are effective to achieve adequatecontrast enhancements. For most MRI applications preferred dosages ofimaging metal ion will be in the range of from 0.02 to 1.2 mmoles/kgbodyweight while for X-ray applications dosages of from 0.05 to 2.0mmoles/kg are generally effective to achieve X-ray attenuation.Preferred dosages for most X-ray applications are from 0.1 to 1.2 mmolesof the lanthanide or heavy metal compound/kg bodyweight. Where thereporter is a radionuclide, dosages of 0.01 to 100 mCi, preferably 0.1to 50 mCi will normally be sufficient per 70 kg bodyweight. Where thereporter is a superparamagnetic particle, the dosage will normally be0.5 to 30 mg Fe/kg bodyweight.

[0354] The dosage of the compounds of the invention for therapeutic usewill depend upon the condition being treated, but in general will be ofthe order of from 1 pmol/kg to 1 mmol/kg bodyweight.

[0355] The compounds of the present invention may be formulated withconventional pharmaceutical or veterinary aids, for example emulsifiers,fatty acid esters, gelling agents, stabilizers, antioxidants, osmolalityadjusting agents, buffers, pH adjusting agents, etc., and may be in aform suitable for parenteral administration, for example injection orinfusion or administration directly into the vasculature. Thus thecompounds of the present invention may be in conventional pharmaceuticaladministration forms such as solutions, suspensions and dispersions inphysiologically acceptable carrier media, for example water forinjections.

[0356] The compounds according to the invention may therefore beformulated for administration using physiologically acceptable carriersor excipients in a manner fully within the skill of the art. Forexample, the compounds, optionally with the addition of pharmaceuticallyacceptable excipients, may be suspended or dissolved in an aqueousmedium, with the resulting solution or suspension then being sterilized.

[0357] Parenterally administrable forms, e.g. intravenous solutions,should be sterile and free from physiologically unacceptable agents, andshould have low osmolality to minimize irritation or other adverseeffects upon administration, and thus the contrast medium shouldpreferably be isotonic or slightly hypertonic. Suitable vehicles includeaqueous vehicles customarily used for administering parenteral solutionssuch as Sodium Chloride Injection, Ringer's Injection, DextroseInjection, Dextrose and Sodium Chloride Injection, Lactated Ringer'sInjection and other solutions such as are described in Remington'sPharmaceutical Sciences, 15th ed., Easton: Mack Publishing Co., pp.1405-1412 and 1461-1487 (1975) and The National Formulary XIV, 14th ed.Washington: American Pharmaceutical Association (1975). The solutionscan contain preservatives, antimicrobial agents, buffers andantioxidants conventionally used for parenteral solutions, excipientsand other additives which are compatible with the chelates and whichwill not interfere with the manufacture, storage or use of products.

[0358] The agents of formula I may be therapeutically effective in thetreatment effective in the treatment of disease states as well asdetectable in in vivo imaging. Thus for example the vector on thereporter moieites may have therapeutic efficacy, eg. by virtue of theradiotherapeutic effect of a radionuclide reporter, the efficacy inphotodynamic therapy of a chromophore (or fluorophore) reporter or thechemotherapeutic effect of the vector moiety.

[0359] Use of the agents of formula I in the manufacture of therapeuticcompositions and in methods of therapeutic or prophylactic treatment ofthe human or non-human animal body are thus considered to representfurther aspects of the invention.

[0360] The present invention will now be further illustrated by way ofthe following non-limiting examples. Unless otherwise indicated, allpercentages given are by weight.

EXAMPLE 1

[0361] Contrast Agent for MR Imaging of Angiogenesis

[0362] Compound 1

[0363] Lysine (0.1 g, 0.7 mmol) is added to a solution ofN²-[3S-hydroxy-4-(N-hydroxyamino)₂R-isobutylsuccinyl]-L-(4-oxymethylcarboxy)phenylalanine-N¹-methylamide (prepared in accordance with WO94/02447,0.3g, 0.7 mmol) and DCC(N,N-dicyclohexylcarbodiimide) in dry DMF(N,N-dimethylformamide). The reaction mixture is stirred at ambienttemperature and is followed by TLC.

[0364] The dispersion is left overnight at +4° C. The dispersion isfiltered and the solvent rotary evaporated before the substance ispurified by chromatography.

[0365] Compound 2

[0366] Diethylenetriaminepentaacetic acid dianhydride (17.9 g, 50 mmol)is dissolved in dry DMF and compound 1 (0.3 g, 0.5 mmol) dissolved indry DMF is added. The reaction mixture is stirred at elevatedtemperature under nitrogen atmosphere. The reaction is followed by TLC.The solvent is rotary evaporated and the substance purified bychromatography.

[0367] Gd(III) Chelate of Compound 2

[0368] To a solution of compound 2 (0.4 g, 0.4 mmol) in water is addedgadolinium oxide Gd2O₃ (0.1 g, 0.2 mmol) and the mixture is heated at95° C. After filtration the solution is evaporated and dried in vacuo at50° C.

EXAMPLE 2

[0369] Contrast Agent for MR Imaging of Angiogenesis

[0370] Compound 3

[0371] Lysine (0.1 g, 0.7 mmol) is added to a solution ofN-(4-octylphenyl)-3-(2-carboxyethyl)-6,7-dihydro-5H-thiazolo[3,2-a]pyrimidine-2-carboxamide(prepared in accordance with EP-A-618208, 0.3 g, 0.7 mmol) and DCC(N,N′-dicyclohexylcarbodiimide) in dry DMF (N,N-dimethylformamide). Thereaction mixture is stirred at ambient temperature and is followed byTLC. The dispersion is left overnight at +4° C. The dispersion isfiltered and the solvent rotary evaporated before the substance ispurified by chromatography.

[0372] Compound 4

[0373] Diethylenetriaminepentaacetic acid dianhydride (17.9 g, 50 mmol)is dissolved in dry DMF and compound 3 (0.3 g, 0.,5 mmol) dissolved indry DMF is added. The reaction mixture is stirred at elevatedtemperature under nitrogen atmosphere. The reaction is followed by TLC.The solvent is rotary evaporated and the substance purified bychromatography.

[0374] Gd(III) Chelate of Compound 4

[0375] To a solution of compound 4 (0.4 g, 0.4 mmol) in water is addedgadolinium oxide Gd₂O₃ (0.1 g, 0.2 mmol) and the mixture is heated at95° C. After filtration the solution is evaporated and dried in vacuo at50° C.

EXAMPLE 3

[0376] Contrast Agent for Nuclear Medicine for Detection of Angiogenesis

[0377]^(99m)Tc Chelate of Compound 2

[0378] Compound 2 from Example 1 (1 mg) is dissolved in 0.1 N NaOH.SnCl₂.2H₂O (100 μg) dissolved in 0.05 N HCl and a solution of 10-100 mCi^(99m)Tc in the form of sodium pertechnetate in saline is added. The pHof the solution is adjusted to pH 7-8 by addition of 0.5 M phosphatebuffer (pH 5) after less than one minute. The reaction is followed byTLC and the substance is purified by chromatography.

EXAMPLE 4

[0379] Contrast Agent for Nuclear Medicine for Detection of Angiogenesis

[0380]^(99m)Tc Chelate of Compound 4

[0381] Compound 4 from Example 2 (1 mg) is dissolved in 0.1 N NaOH.SnCl₂.2H₂O (100 μg) dissolved in 0.05 N HCl and a solution of 10-100 mCi^(99m)Tc in the form of sodium pertechnetate in saline is added. The pHof the solution is adjusted to pH 7-8 by addition of 0.5 M phosphatebuffer (pH 5) after less than one minute. The reaction is followed byTLC and the substance is purified by chromatography.

EXAMPLE 5

[0382] Contrast Agent for Nuclear Medicine for Detection of Angiogenesis

[0383] An aqueous solution of ¹³¹I₂(2 equivalents) and sodiumperchlorate (1 equivalent) is added to an aqueous solution ofN²-[3S-hydroxy-4-hydroxyamino)-2R-isobutylsuccinyl]-L-phenylalanine-N′-methylamide(prepared in accordance with WO94/02446, 1 equivalent). The solvent isrotary evaporated and the substance is purified by chromatography.

EXAMPLE 6

[0384] Preparation of a DTPA monoamide gadolinium complex comprising avector for targeting of VEGF receptor for MR detection of angiogenesis

[0385] (Gd complex)

[0386] a) Synthesis of 6,7-dimethoxy-3H-quinazolin-4-one

[0387] A mixture of 2-amino-4,5-dimethoxybenzoic acid (9.9 mg, 0.050mmol) and formamide (5 ml) was heated at 190° C. for 6 hours. Themixture was cooled to 80° C. and poured onto water (25 ml). Precipitatedmaterial was filtered off, washed with water and dried in vacuo. Yield1.54 g (15%), brown powder. The structure was confirmed by ¹H (500 MHz)and ¹³C NMR (125 MHz) analysis.

[0388] b) Synthesis of 4-chloro-6.7-dimethoxyquinazoline

[0389] A suspension of compound from a) (1.03 g, 5.00 mmol) inphosphorousoxychloride (20 ml) was refluxed for 3 hours. The darksolution was concentrated and the residue was taken up in ethyl acetate.The organic phase was washed with saturated sodium bicarbonate solutionand dried (MgSO₄). The solution was filtered through a short silicacolumn and concentrated to give 392 mg (35%) of off-white material. ¹HNMR (300 MHz) and ¹³C NMR (75 MHz) spectra were in accordance with thestructure.

[0390] c) Synthesis of[4-(6.7-dimethoxy-quinazolin-4-ylamino)-phenyl]acetic Acid

[0391] A mixture of compound from b) (112 mg, 0.500 mmol) and4-aminophenylacetic acid (76 mg, 0.50 mmol) in 2-propanol (8 ml) wasrefluxed for 3 hours. The reaction mixture was cooled and precipitatedmaterial was isolated, washed with 2-propanol and dried in vacuo. Yield183 mg (97%), pale yellow solid material. The structure was verified by¹H NMR (500 MHz) and ¹³C NMR (125 MHz) analysis. Furthercharacterisation was carried out using MALDI mass spectrometry(α-cyano-4-hydroxycinnamic acid matrix), giving m/z for [MH]⁺ at 341,expected 340.

[0392] d) Synthesis of t-butyl(6-{2-[4-(6,7-dimethoxy-quinazolin-4-ylamino)phenyl]acetylamino}hexyl)carbamate

[0393] To a suspension of compound from c) (38 mg, 0.10 mmol) andN-Boc-1,6-diaminohexane hydrochloride (25 mg, 0.10 mmol) in DMF (2.0 ml)was added N,N-diisopropylethyl-amine (34 ml, 0.20 mmol). To the clearsolution was added N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidhydrochloride (19 mg, 0.10 mmo) and 1-hydroxybenzo-triazole (15 mg, 0.10mmol). The reaction mixture was stirred at room temperature overnightand then poured onto 25 ml of water containing sodium carbonate (2.5 g)and sodium chloride (4.0 g). Organic material was extracted intochloroform and the organic phase was ashed with water and dried(Na₂SO₄). The solution was filtered and concentrated. The product waspurified by column chromatography (silica, chloroform/methanol/aceticacid 85:10:5) and finally lyophilised from acetic acid. Yield 54 mg(90%), yellow-white solid material (acetate). The product wascharacterised by MALDI mass spectrometry (a-cyano-4-hydroxycinnamic acidmatrix), giving m/z for [MH]⁺ at 539 as expected. Furthercharacterisation was carried out using ¹H (500 MHz) and ¹³C (125 MHz)NMR spectroscopy.

[0394] e) Synthesis ofN-(6-aminohexyl)-[4-(6,7-dimethoxy-quinazolin-4-ylamino)phenyl]acetamideHydrochloride

[0395] Compound from d) (27 mg, 0.050 mmol) was dissolved in dioxane (3ml) by gentle heating. To the solution was added 4 N HCl in dioxane (0.5ml). The reaction mixture was stirred overnight and concentrated invacuo to give a quantitative yield of the title compound.Characterisation was carried out using MALDI mass spectrometry(a-cyano-4-hydroxycinnamic acid matrix), giving m/z for [MH]⁺ at 439 asexpected. Further characterisation was carried out using analytical HPLC(column Vydac 218TP54, gradient 12-24% B over 20 min, A=water/0.1% TFA,B=acetonitrile/0.1% TFA, flow rate 1.0 ml/min) giving a single productpeak with retention time 13.0 min detected at 340 nm. Characterisationwas also carried out by means of NMR spectroscopy, giving ¹H (500 MHz)and ¹³C (125 MHz) spectra in accordance with the structure.

[0396] f) Synthesis of a DTPA Monoamide Derivative for GadoliniumChelation (Structure Shown Above)

[0397] N,N-Diisopropylethylamine (17 μl, 0.10 mmol) was added to asuspension of compound from e) (0.05 mmol) and DTPA-anhydride (179 mg,0.500 mmol) in DMF (5 ml). The mixture was stirred at room temperaturefor 2 hours and concentrated in vacuo. HPLC analysis (column Vydac218TP54, gradient 16-28% B over 20 minutes, A=water/0.1% TFA,B=acetonitrile/0.1% TFA, flow rate 1.0 ml/min) gave a product peak at7.9 min shown by LC-MS (ESI) to correspond to the title compound (m/zfor [MH]⁺ at 813, expected 814). The product was purified by preparativeHPLC (column Vydac 218TP1022, gradient 16-28% B over 60 min, Awater/0.1% TFA, B=acetonitrile/0.1% TFA, flow rate 10.0 ml/min,detection at 254 nm) giving a yield of 6.7 mg of purified material.Analytical HPLC analysis of purified material showed a shift inretention time to 5.6 min (analytical conditions as described above),shown by MALDI mass spectrometry to correspond to the iron complex,giving m/z at 870 for the complex and 816 for the free ligand.

[0398] g) Preparation of the Gadolinium Complex of Compound from f)

[0399] Compound from f) (0.1 mg) was dissolved in an aqueous solution ofgadolinium trichloride (conc 2 mg/ml, 0.1 ml). The mixture was stirredovernight. Quantitative conversion to the gadolinium complex wasverified by MALDI mass spectrometry (α-cyano-4-hydroxycinnamic acidmatrix), giving m/z peaks at 970, 992 and 1014 for the gadoliniumcomplex (gadolinium, gadolinium/sodium and gadolinium/disodium,respectively) and at 816/838 corresponding to the free ligand/sodiumcomplex. No trace of the iron complex could be detected.

EXAMPLE 7

[0400] Preparation of a DTPA Bisamide Gadolinium Complex Comprising aVector for Targeting of VEGF Receptor for MR Detection of Angiogenesis

[0401] (Gd complex)

[0402] a) Synthesis of a DTPA Bisamide Derivative for GadoliniumChelation (Structure Shown Above)

[0403] Analytical HPLC of the reaction mixture in Example 6f) gave alsoa peak at 16.8 min that was shown by LC-MS (EIS) analysis to correspondto the DTPA bisamide shown above, giving m/z at 1233 for [MH]⁺ asexpected and 616.6 as expected for [MH2]²⁺. The product was purified bypreparative HPLC (conditions as described in Example 6f)) to give 14 mgof pure material after lyophilisation. Analytical HPLC analysis of thepurified material showed a shift in retention time from 16.8 min (in thecrude mixture) to 11.1 min due to formation of the iron complex duringpurification, as verified by MALDI mass spectrometry(≢-cyano-4-hydroxy-cinnamic acid matrix) giving m/z at 1291 for the ironcomplex and 1237 for the free ligand.

[0404] b) Preparation of the Gadolinium Complex of Compound from a)

[0405] The compound from a) was treated with an of gadoliniumtrichloride as described in Example 6g). After 2 hours reaction timeMALDI mass spectrometry showed conversion to the gadolinium complex,giving m/z at 1391 for the gadolinium complex and 1235 for the freeligand.

Example 8

[0406]Carboxymethyl-[2-(carboxymethyl-{2-[carboxymethyl-({2-[3-({3-oxo-2-[2-(pyridin-2-ylamino)-ethyl]-2,3-dihydro-1H-isoindole-5-carbonyl}-amino)-propionylamino]-ethylcarbamoyl}-methyl)-amino]-ethyl}-amino)-ethyl]-amino}-aceticAcid (12)

[0407] a) 4-Methyl-Isophthalic Acid (2)

[0408] To THF (130 ml) solution of 3-bromo-4-methyl-benzoic acid (5.0g,23.25 mmol) under nitrogen and cooled to −78° C. (dryice/methanol) wasadded MeMgBr in ether (3.0 M, 8.5 ml, 25.57 mmol) at such a rate thatthe temperature did not exceed −75° C. The temperature was then allowedto rise to −60° C. and after gas evolution had ceased, the solution wascooled again to −78° C. BuLi in hexane (1.6 M, 29.06 ml, 46.50 mmol) wasthen added dropwise such that the temperature did not rise above −75° C.The mixture was then stirred at this temperature for 15 minutes beforecrushed dryice (4.4 g, 100 mmol) was added. The precipitate wasvigorously stirred as the temperature was allowed to freely rise toambient temperature. The mixture was made acidic using 6 N HCl and thesolid material collected filteration, washed with diethyl ether anddried. Recrystallisation from water afforded off-white pure compound(81%) m.p. 296-298° C. (sublimed). NMR conforms to expected structure.

[0409] b) 4-Methyl-isophthalic Methyl Ester (4)

[0410] A mixture of compound (2) (2.83 g, 15.71 mmol), thionyl chloride(50 ml) and DMF (3 drops) was heated at reflux for 2 hours. Aftercooling to room temperature, excess thionyl chloride was removed underreduced pressure (rotary evaporator). The dark oil which was obtainedwas dissolved in carbon tetrachloride (30 ml), treated with pyridine (1ml, 12.43 mmol) and methanol (20 ml) and stirred at ambient temperaturefor 2 hours. The solvents were evaporated and the residue purified byflash chromatography: silica, hexane/EtOAc (9:1).

[0411] c) 4-Bromoethyl-isophthalic acid dimethylester (5)

[0412] A mixture of (4) (0.96 g, 4.61 mmol), dibenzoylperoxide (56 mg,0.23 mmol) and N-bromosuccinimide (NBS) (0.829, 4.61 mmol) in carbontetrachloride (20 ml) was heated at reflux for 20 hours. After coolingto room temperature and filteration the solvent was evaporated to give ayellow oil. Flash chromatograph: silica, hexane/EtOAc (7:3) afforded thepure compound.

[0413] d)3-oxo-2-[2-(pyridin-2-ylamino)-ethyl]2,3-dihydro-1H-isoindole-5-carboxylicAcid Methyl Ester (6)

[0414] A solution of (5) (511 mg, 1.78 mmol) in toluene (10 ml) wastreated with Et₃N (744 μ1,5.33 mmol) andN1-pyridin-2-yl-ethane-1,2-diamine (244 mg, 1.78 mmol) [prepared bytreatment of 2-bromopyridine with excess ethylene diamine and pyridine]and the mixture refluxed for 6 hours. After cooling to room temperatureand evaporation of the solvent, the residue was purified by flashchromatography: silica, CH₂Cl₂/acetone (3:2).

[0415] e)3-oxo-2-[2-pyridin-2-ylamino)-ethyl]2,3-dihydro-1H-isoindole-5-carboxylicAcid (7)

[0416] A methanol solutin (6 ml) of (6) (301 mg, 0.97 mmol) and 1 N NaOH(3 ml) was stirred at ambient temperature for 24 hours. The solution wasmade acidic using 1 M NaHSO₄ solution and the precipitated product wascollected by filteration washed thoroughly with water and dried overP₂O₅/bluegel for 24 hours.

[0417] f)3-({3-oxo-2-[2-(pyridin-2-ylamino)-ethyl]2,3-dihydro-1H-isoindole-5-carbonyl}-amino)-propionicAcid tert-butyl Ester (8)

[0418] A solution of (7) (166 mg, 0.56 mmol), N-methyl morfolin (185 μl,1.68 mmol), BOP (322 mg, 0.73 mmol) and H-β-ala-OtBu (152 mg, 0.84 mmol)in DMF (5 ml) was stirred at ambient temperature for 20 hours. Themixture was diluted with ethyl acetate (10 ml) and then washed once eachwith H₂O, NaHCO₃, 10% KHSO₄ and brine (5 ml), dried (MgSO₄) andconcentrated. Flash chromatography (silica, EtOAc) gave the ester (8) asa white solid.

[0419] g)3-({3-oxo-2-[2-(pyridin-2-ylamino)-ethyll2.3-dihydro-1H-isoindole-5-carbonyl}-amino)-propionicAcid (9)

[0420] A solution of the ester (8) (252 mg, 0.60 mmol), TFA (4 ml) andCH₂Cl₂ (8 ml) was stirred at ambient temperature for 3 hours. Themixture was evaporated to dryness and the residue purified by flashchromatography (silica, EtOH/NH₄OH 19:1) to provide (9) as off-whitefoam. NMR conforms to structure.

[0421] h){2-[3-({3-Oxo-2-[2-(pyridin-2-ylamino)-ethyl]-2,3-dihydro-1H-isoindole-5-carbonyl-amino)-propionylamino]-ethyl}-carbamicAcid Tert-Butyl Ester (10)

[0422] To a solution of the acid (9) (20 mg, 0.054 mmol) in DMF (2 ml)was added N-methyl morfolin (NMM) (16.40 mg, 0.162 mmol), BOP (castro'sreagent) 31.05 mg, 0.070 mmol) and the BOC-protected diamine (13 mg,0.081 mmol) and the mixture was stirred at ambient temperature for 20hours. After dilution with EtOAc (5 ml), the solution was washed onceeach with H₂O, sat. NaHCO₃, 10% KHSO₄ and brine. The organic phase wasdried (MgSO₄) and concentrated. Flash chromatography (silica, 1:1CH₂Cl₂/acetone) MALDI-MS; 510.59.

[0423] i)3-Oxo-2-[2-(pyridin-2-ylamino)-ethyl]-2,3-dihydro-1H-isoindole-5-carboxylicAcid [2-(2-amino-ethylcarbamoyl)-ethyl]-amide (11)

[0424] A CH₂Cl₂ solution (3 ml) of (10) (20 mg, 0.039 mmol) and TFA (2ml) was stirred under ambient conditions for 4 hours. The reactionmixture was concentrated and the residue purified by flashchromatography (silica, 19:1, CH₂Cl₂/acetone) to provide (4) as a whitesolid. MALDI-MS; 410.48

[0425] j)Carboxymethyl-[2-(carboxymethyl-(2-[carboxymethyl-({2-[3-({3-oxo-2-[2-(pyridin-2-ylamino)-ethyl]-2,3-dihydro-1H-isoindole-5-carbonyl}-amino)-propionylamino]-ethylcarbamoyl}-methyl)-amino]-ethyl}-amino)-ethyl]-amino}-aceticAcid (12)

[0426] A solution of (11) (15 mg, 0.36 mmol), N,N-diisopropylamine (17μl, 0.10 mmol) and DTPA-anhydride (129 mg, 0.36 mmol) in DMF (2 ml) wasstirred at ambient temperature for 3 hours and concentrated in vacuo.The residue was purified by preparative HPLC (acetonirile/0.1% TFA inwater).

EXAMPLE 9

[0427] Preparation of a DTPA Monoamide Gadolinium Complex Comprising aVector for Targeting of bFGF Receptor for MR Detection of Angiogenesis

[0428] a) Synthesis of:

[0429]{3-[2-(4-chloro-benzyloxy)-2-(2.4-dichloro-phenyl)-ethyl]-3H-imidazol-1-yl}-aceticAcid Tert-Butyl Ester

[0430]1-[2-(4-chloro-benzyloxy)-2-(2,4-dichloro-phenyl)-ethyl]-1H-imidazole (1g, 2.25 mmol) and tert-butyl bromoacetate (1 ml, 6.8 mmol) weredissolved in 15 ml acetonitrile and heated to reflux overnight. TLCshowed full conversion of the starting material. The solvent was cooled,evaporated in vacuo, and the residual oil was dissolved in chloroformand triturated with ether. The product was identified by Maldi massspectrometry, and used in the next step without further purification.

[0431] b) Synthesis of:

[0432]{3-[2-(4-chloro-benzyloxy)-2-(2.4-dichloro-phenyl)-ethyl]-3H-imidazol-1-yl}-aceticAcid

[0433] The compound from a) (500 mg, 1 mmol) was dissolved in 2 mldichloromethane and cooled in an icebath. 2 ml trifluoroacetic acid wasadded, the icebath was removed and the reaction mixture was stirred for1 hour. TLC showed full conversion of the starting material. The solventwas removed in vacuo, and the product used in the next step withoutfurther purification.

[0434] c) Synthesis of:

[0435][6-(2-{3-[2-(4-chloro-benzyloxy)-2-(2,4-dichloro-phenyl)-ethyl]-3H-imidazol-1-yl}-acetylamino)-hexyl]-carbamicAcid Tert-Butyl Ester

[0436] The product from b) was converted to c) by the proceduredescribed for Example 6 d), and the product was purified by flashchromatography.

[0437] d) Synthesis of:

[0438]N-(6-amino-hexyl)-2-{3-[2-(4-chloro-benzyloxy)-2-(2,4-dichloro-phenyl)-ethyl]-3H-imidazol-1-yl}-acetamide

[0439] The product from c) was converted to d) by the proceduredescribed for Example 6 e). The product was used without furtherpurification.

[0440] e) Synthesis of:

[0441]((2-{[2-(bis-carboxymethyl-amino)-ethyl]-carboxymethyl-amino]-ethyl)-}[6-(2-{3-[2-(4-chloro-benzyloxy)-2-(2,4-dichloro-phenyl)-ethyl]-3H-imidazol-1-yl}-acetylamino)-hexylcarbamoyl]-methyl}-amino)-aceticAcid

[0442] The product from d) was converted to e) by the proceduredescribed for Example 6 f). Purification was performed by preparativeHPLC as described in Example 6 f).

[0443] f) Preparation of the gadolinium complex of compound e) wasperformed by the procedure described in Example 6 g).

1 5 1 16 PRT Artificial Sequence UNSURE (10)..(10) Xaa represents anunidentified amino acid 1 Leu Pro Ala Leu Pro Glu Asp Gly Gly Xaa GlyAla Phe Pro Pro Gly 1 5 10 15 2 17 PRT Artificial Sequence SITE (1)..(1)Xaa represents Ala or Ser 2 Xaa Xaa Pro Xaa Xaa Ala Gly Thr Met Ala AlaGly Ser Ile Thr Thr 1 5 10 15 Leu 3 54 PRT Artificial Sequence SITE(1)..(54) A modified platelet factor 4 3 Ser Gln Val Arg Pro Arg His IleThr Ser Leu Glu Val Ile Lys Ala 1 5 10 15 Gly Pro His Cys Pro Thr AlaGln Leu Ile Ala Thr Leu Lys Asn Gly 20 25 30 Arg Lys Ile Cys Leu Asp LeuGln Ala Pro Leu Tyr Lys Lys Ile Ile 35 40 45 Lys Lys Leu Leu Glu Ser 504 53 PRT Artificial Sequence SITE (1)..(53) A modified platelet factor 44 Ser Gln Val Arg Pro Arg His Ile Thr Ser Leu Glu Val Ile Lys Ala 1 5 1015 Gly Pro His Cys Pro Thr Ala Gln Leu Ile Ala Thr Leu Lys Asn Gly 20 2530 Arg Lys Ile Cys Leu Asp Leu Gln Ala Pro Leu Tyr Lys Ile Ile Lys 35 4045 Lys Leu Leu Glu Ser 50 5 16 PRT Artificial Sequence SITE (1)..(16)Modified platelet factor 4 5 Glu Ala Glu Glu Asp Gly Asp Leu Gln Cys LeuCys Val Lys Thr Thr 1 5 10 15

1. A composition of matter of formula I V-L-R  (I) where v is a vectormoiety having affinity for an angiogenesis-related endothelial cellreceptor, l is a linker moiety or a bond and r is a detectable moiety,characterised in that v is a non-peptidic organic group, or v ispeptidic and r is a macromolecular or particulate species providing amultiplicity of labels detectable in in vivo imaging.
 2. A compositionof matter as claimed in claim 1 wherein V is a non-peptidic organicmoiety.
 3. A composition of matter as claimed in claim 1 wherein V is apeptide or peptoid moiety.
 4. A composition of matter as claimed inclaim 1 wherein V is a vector for integrin receptor.
 5. A composition ofmatter as claimed in claim 1 wherein V is a vector for fibronectinreceptor.
 6. A composition of matter as claimed in claim 1 wherein V isa vector for the VEGF receptor.
 7. A composition of matter as claimed inclaim 1 wherein V is a vector for the urokinase plasminogen activatorreceptor (UPAR).
 8. A composition of matter as claimed in claim 1wherein V is selected from the list of vector moieties comprising:N²-[3S-hydroxy-4-(N-hydroxyamino)2R-isobutylsuccinyl]-L-(4-oxymethylcarboxy)phenylalanine-N¹-methylamide,N-(4-octylphenyl)-3-(2-carboxyethyl)-6,7-dihydro-5H-thiazolo[3,2-a]pyrimidine-2-carboxamide,N²-[3S-hydroxy-4-hydroxyamino)-2R-isobutylsuccinyl]-L-phenylalanine-N′-methylamide,N-(6-aminohexyl)-[4-(6,7-dimethoxy-quinazolin-4-ylamino)phenyl]acetamidehydrochloride,3-Oxo-2-[2-(pyridin-2-ylamino)-ethyl]-2,3-dihydro-1H-isoindole-5-carboxylicacid [2-(2-amino-ethylcarbamoyl)-ethyl]-amide, andN-(6-amlno-hexyl)-2-{3-[2-(4-chloro-benzyloxy)-2-(2,4-dichloro-phenyl)-ethyl]-3H-imidazol-1-yl}-acetamide.9. A composition of matter as claimed in claim 1 wherein V-L-R isselected from the following residues: Gd(III), ^(99m)Tc or ¹¹¹Inchelates of the DTPA derivatives of the vector moieties as claimed inclaim
 8. 10. A composition of matter as claimed in any one of claims 1to 8 wherein R is a radionuclide.
 11. A composition of matter as claimedin claim 10 wherein R is an iodine or metal radionuclide.
 12. Apharmaceutical composition comprising a composition of matter of formulaI as defined in any one of claims 1 to 11 together with at least onepharmaceutically acceptable carrier or excipient.
 13. The use of acomposition of matter of formula I as defined in any one of claims 1 to11 for the manufacture of a contrast medium for use in a method ofdiagnosis involving administration of said contrast medium to an animatesubject and generation of an image of at least part of said subject. 14.A method of generating an image of an animate human or non-human animalsubject involving administering a contrast agent to said subject andgenerating an image of at least a part of said subject to which saidcontrast agent has distributed, characterised in that as said contrastagent is used a composition of matter of formula I as defined in any oneof claims 1 to
 11. 15. A method of monitoring the effect of treatment ofa human or non-human animal subject with a drug to combat or provokeeffects associated with angiogenesis, said method involvingadministering to said subject a composition of matter of formula I asdefined in any one of claims 1 to 11 and detecting the uptake of saidagent by angiogenesis related endothelial cell receptors, saidadministration and detection optionally but preferably being effectedrepeatedly, eg. before, during and after treatment with said drug.
 16. Aprocess for the preparation of a composition of a matter of formula I asdefined in any one of claims 1 to 11, said process comprisingconjugating (i) an organic compound having binding affinity for anendothelial cell receptor associated with angiogenesis to (ii) acompound detectable in a diagnostic imaging procedure or a chelantcompound and if necessary metallating chelant groups in the resultantconjugate with a metal ion detectable in a diagnostic imaging procedure.